Immunoadhesin comprising a glycorprotein v1 domain

ABSTRACT

The present invention provides a fusion protein comprising (a) the extracellular domain of glycoprotein VI or a variant thereof that is functional for binding to collagen and (b) the Fc domain of an immunoglobulin or a function-conservative part thereof, characterised by a polypeptide chain having an amino acid sequence as shown in FIG.  7  and whereby the fusion protein is obtainable by a process which provides the fusion protein in the form of a specific dimer.

The present invention relates to an immunoadhesin comprising a specificglycoprotein VI domain. The immunoadhesin of the invention is obtainableby a specific process providing the immunoadhesin in the form of adimer. The present invention also relates to the use of theimmunoadhesin of glycoprotein VI for the preparation of a medicament forthe prevention of intraarterial thrombosis in a specific group ofpatients. Moreover, the present invention relates to the the use of theimmunoadhesin of glycoprotein VI for the preparation of a medicament forthe prevention and treatment of atheroprogression. The present inventionalso relates to the use of the immunoadhesin of glycoprotein VI for thepreparation of a medicament for the prevention and treatment of chronicprogression of atherosclerosis in diabetic patients. The presentinvention also relates to in vitro and in vivo screening methods for anInhibitor of GPVI mediated adhesion of platelets to active intravascularlesions.

Acute coronary or carotid syndromes are a major cause of death inWestern societies. Even in case of an initial survival of such acardiovascular event, many patients suffer from life-threateningcomplications such as intravascular thrombosis leading to furthermyocardial infarction or stroke.

Intravascular thrombosis is the result of aggregation of platelets in avessel whereby the blood flow in the vessel may be seriously reduced oreven completely inhibited. Specifically, the disruption of anatherosclerotic plaque initiates a cascade of events culminating inarterial thrombosis and ischemia of the downstream tissue, precipitatingdiseases such as myocardial infarction or ischemic stroke. The firstresponse to vascular injury is adhesion of circulating platelets toexposed subendothelial matrix proteins, which triggers subsequentplatelet aggregation. Among the macromolecular components of thesubendothelial layer fibrillar collagen is considered the mostthrombogenic constituent, as it acts as a strong activator of plateletsand supports platelet adhesion both in vitro and in vivo (1-3).

The platelet membrane proteins, which have been reported to be putativecollagen receptors, may be divided into those which interact indirectlywith collagen through collagen-bound von Willebrand factor (vWf),including GPIbα and the integrin α_(IIb)β₃, and those which interactdirectly with collagen including GPVI, the integrin α₂β₁, and CD36(reviewed in (2)). Only recently, the platelet glycoprotein VI (GPVI)has been identified as the major platelet collagen receptor (4). GPVI isa 60-65 kDa type I transmembrane glycoprotein, which belongs to theimmunoglobulin superfamily (5;6). In human and mouse platelets GPVIforms a complex with the FcR γ-chain at the cell surface (7;8). Ligandbinding to GPVI triggers tyrosine phosphorylation of the ITAM motif ofthe Fc receptor g chain initiating downstream signaling via Syk kinases,LAT, SLP-76, and phospholipase C (9-13). Platelets deficient in GPVIshow loss of collagen-induced adhesion and aggregation in vitro (4;14).Likewise, function blocking anti-GPVI monoclonal antibodies attenuate exvivo platelet aggregation in response to collagen and collagen-relatedpeptide CRP, which mimics collagen triple helix (15;16).

It is known that the problem of complications due to the aggregation ofplatelets can be addressed by administering inhibitors of plateletaggregation. For the treatment of acute coronary syndromes, GP IIb/IIIainhibitors such as ReoPro significantly improve the outcome of patients.However, a recent meta-analysis of clinical trials revealed asignificant remaining risk for death or myocardial infarction despiteoptimal antithrombotic intervention (Boersma E, Harrington R A,Moliterno D J, White H, Therouxi P, Van de Werf F, de Torbal A,Armstrong P W, Wallentin L C, Wilcox R G, Simes J, Califf R M, Topol EJ, Simoons M L. Platelet glycoprotein IIb/IIIa inhibitors in acutecoronary syndromes: a meta-analysis of all major randomised clinicaltrials. Lancet 2002; 359:189-98). Specific severe side effects of thistherapeutic regimen are bleeding complications. These occurred in 2.4%of the patients with the most severe form of intracranial bleedingoccuring in almost 0.1% of the treated patients. Several mechanisticshortcomings of the GP IIb/IIIa receptor blockade have been revealedwhich account for suboptimal effectivity and side effects (Dickfeld T,Ruf A, Pogatsa-Murray G, Muller I, Engelmann B, Taubitz W, Fischer J,Meier 0, Gawaz M. Differential antiplatelet effects of variousglycoprotein IIb-IIIa antagonists. Thromb Res. 2001;101:53-64. Gawaz M,Neumann F J, Schomig A. Evaluation of platelet membrane glycoproteins incoronary artery disease: consequences for diagnosis and therapy.Circulation. 1999;99:E1-E11).

The inhibition of platelet aggregation leads to a general impairment ofthe platelets with regard to their ability to aggregate. Accordingly,not only the undesired thrombosis formation is influenced, but also thegeneral ability of the platelets to terminate bleeding. Therefore, theadministration of inhibitors of platelet aggregation inherently leads tosevere side effects such as bleedings which may cause furtherlife-threatening complications. These side effects are of course stillmore problematic in patients suffering from diabetes.

Diabetes is one of the main risk factors for atherosclerosis.Additionally diabetes constitutes an increased risk of life threateningcomplications and excess morbidity in patients presenting with acutevascular and especially coronary syndromes. Diabetic patients withunstable angina present with a higher incidence of plaque ulceration andintracoronary thrombosis compared to non-diabetic patients.(Biondo-Zoccai GGL; Abbate A; Liuzzo G, Biasucci L: Atherothrombosis,inflammation, and diabetes. J Am Coll Cardiol 41; 1071-1077; 2003).

It is increasingly recognized that platelets are a major trigger for theprogression of atherosclerosis. The link between increasedatheroprogression, and increased platelet responsiveness and diabetes isso far an unresolved problem. Diabetic patients suffer from acutevascular complications independent of the degree of atherosclerosisindicative of different presently unknown mechanisms for plateletactivation in the development of diabetic acute vascular complicationsand atherosclerotic acute vascular complications.

Therefore, it is the problem of the invention to provide a medicamentwhich is useful for avoiding life-threatening complications subsequentto an acute coronary or carotid syndrome while maintaining the potencyof the blood for hemostasis.

It is a further problem of the preent invention to provide a medicamentfor the treatment or prevention of atheroprogression.

It is a still further problem of the invention to provide a medicamentfor the treatment of diabetes, notably complications associated withdiabetes.

It is a further problem of the invention to provide an in vitro and anin vivo screening method for inhibitors of adhesion of platelets tointravascular lesions.

GENERAL DESCRIPTION OF THE INVENTION

The above problems are solved according to the claims. The presentinvention provides the first direct in vivo evidence indicating thatGPVI is in fact strictly required in the process of platelet recruitmentunder physiological shear stress following vascular injury. In differentmouse models of endothelial denudation both inhibition or absence ofGPVI virtually abolished platelet-vessel wall interactions and plateletaggregation, identifying GPVI as the major determinant of arterialthrombus formation. This indicates that inhibition of GPVI-ligandinteractions prevents arterial thrombosis in the setting ofatherosclerosis. The present invention uses the antithrombotic potentialof a specific soluble form of GPVI. Specifically, a fusion protein isprovided, which contains the extracellular domain of GPVI and a humanN-terminal Fc tag. The soluble form of human GPVI specifically binds tocollagen with high affinity and attenuated platelet adhesion toimmobilized collagen in vitro and to sites of vascular injury in vivo.Accordingly, the present invention is based on the recognition that theprecondition for intraarterial thrombosis as an acute clinicalcomplication is the initial adhesion of platelets to active lesions inthe vessel walls. The present inventors have recognised that plateletadhesion to subendothelial matrix collagen at a lesion of the vesselwall by the glycoprotein VI (GPVI) receptor represents the key event forthe formation of thrombosis. The inhibition of the adhesion of plateletsto subendothelial matrix collagen of the fusion protein of the inventionis therefore capable of not only preventing adhesion of platelets to anactive lesion, but also to prevent aggregation of platelets at theactive lesion. Thereby, the formation of intravascular thrombosis can beefficiently avoided without impairing the general ability of theplatelets for aggregation.

It is surprising that the complex process of the formation of thrombosismay be inhibited by the inhibition of a single platelet receptor in viewof the fact that different components of the subendothelial layers areligands and activators of platelets such as laminin, fibronectin, vonWillebrand factor (vWf) and collagen. Moreover, a wide variety ofreceptors on the platelets had been proposed by in vitro examinations,but the relevant receptor or receptor combinations which influenceadhesion of platelets to lesions in vivo had not been known before.

The present invention is also based on the recognition that GP VI is amajor meditor of platelet activity for the progression ofatherosclerosis. It is demonstrated that inhibition of thecollagen-medited GPVI activation attentuates atheroprogression inatherosclerosis prone Apo e −/− mice (see FIG. 16). Moreover, it isdemonstrated that the platelets from diabetic patients, who are alsoprone for advanced atherosclerosis and increased thromboticcomplications show an increaed expression of the GPVI-coreceptorFc-receptor. Therefore platelets from diabetics might show increasedresponsiveness to collagen stimulation leading to the clinicallyobserved increased thrombotic complications in unstable angina, wherecollagen is uncovered from subendothelial vascular layers by plaquerupture or endothelial denudation.

The present invention provides therefore a treatment ofatheroprogression in patients, notably in patients suffering fromdiabetes. Moreover, the invention provides a medicament for thetreatment of acute vascular complications such as intravascularthrombosis especially in patients with diabetes. The immunoadhesinFc-GPVI-nt is a potent therapeutic tool to attenunate atheroprogressionand increased responsiveness of platelets to collagen via the GPVIreceptor. Therefore, Fc-GPVI-nt is a medicament for treatment ofatherosclerosis and particularly for the treatment of atheroscleroticcomplications in diabetes.

This invention provides a fusion protein (Fc-GPVI-nt) comprising thefollowing segments:

-   (a) the extracellular domain of glycoprotein VI (GP VI) or a variant    thereof that is functional for binding to collagen and-   (b) the Fc domain of an immunoglobulin or a function-conservative    part thereof.

The fusion protein is characterised by an amino acid sequence as shownin FIG. 7. The fusion protein according to the invention is obtained orobtainable by

-   (a) collecting 2 days after infection the culture supernatant of    Hela cells infected with an adenovirus for Fc-GPVI-nt coding for an    amino acid sequence as shown in FIG. 7;-   (b) centrifuging (3800 g, 30 min, 4° C.) the supernatant of step    (a);-   (c) filtrating (0.45 μm) the supernatant of step (b);-   (d) precipitating the immunoadhesin by addition of 1 vol. ammonium    sulfate (761 g/l) and stirring overnight at 4° C.;-   (e) pelletizing the proteins by centrifugation (3000 g, 30 min, 4°    C.),-   (f) dissolving the pelletized proteins of step (e) in 0.1 Vol PBS    and dialysed in PBS overnight at 4° C.;-   (g) clarifying the protein solution by centrifugation (3000 g, 30    min, 4° C.);-   (h) loading the solution of step (g) on a protein A column (HiTrapυ    protein A HP, Amersham Pharmacia Biotech AB, Uppsala, Sweden);-   (i) washing the column with binding buffer (20 mM sodium phoshate    buffer pH 7.0, 0.02% NaN₃) until OD₂₈₀<0.01;-   (k) eluting fractions with elution buffer (100 mM glycine pH 2.7);-   (I) neutralizing the eluted fractions with neutralisation buffer (1    M Tris/HCl pH 9.0, 0.02% NaN₃);-   (m) pooling the fractions;-   (n) dialysing the pooled fractions in PBS overnight at 4° C.,-   (O) aliquoting the dialysed product and freezing at −20° C.

Under the above conditions, the fusion protein is obtained as acovalently linked dimer of a molecular mass of 160 kDa as measured undernon-reducing conditions by SDS-PAGE. Dimerisation of the fusion proteinpresumably occurs by inter-chain disulfide bonds of cysteins in aspecific domain adjacent to the GPVI fragment of the amino acid sequenceas shown in FIG. 7. The dimeric nature of the fusion protein depends atleast from the presence of a specific region between the Fc portion andthe GPVI portion as contained in FIG. 7, and the preparation process. Amonomeric fusion protein is not useful as a therapeutic agent inpractice since the inferior binding properties of a monomeric fusionprotein as compared to the dimeric fusion protein would requireadministration of protein in an amount which is in the order of onemagnitude larger than the amount of the dimeric fusion protein forobtaining a similar effect, cf. FIG. 9(e). The administration of largeamounts of protein is, however, problematic from a therapeuic andeconomic point of view, in particular in the treatment of chronicdisease.

The fusion protein of the invention is an immunoadhesin. It has asegment (a) that has the function of the extracellular domain ofplatelet GPVI. Said GPVI may be a mammalian GPVI, preferably it is humanGPVI. Said function is preferably binding to the GP VI ligand collagen.The whole extracellular domain of GPVI may be used for said fusionprotein or any fragments thereof provided said fragments are capable ofbinding to collagen. A variant of the fusion protein may have amodification at one or several amino acids of said fusion protein (e.g.glycosylation, phosphorylation, acetylation, disulfide bond formation,biotinylation, chromogenic labelling like fluorescein labelling etc.).Preferably, a variant is a homolog of said fusion protein. An engineeredvariant may be tested easily for its capability of binding to collagenusing the methods disclosed herein. Most preferably, the polypeptide ofresidues 1 to 267 of SEQ ID No: 1 is used as segment (a). However, saidpolypeptide may also be modified by exchanging selected amino acids orby truncating said sequence without abolishing said function.

Segment (b) of said fusion protein serves at least one of the followingpurposes: secretion of the fusion protein from cells that produce saidfusion protein, providing segment (a) in a form (e.g. folding oraggregation state) functional for binding collagen, affinitypurification of said fusion protein, recognition of the fusion proteinby an antibody, providing favourable properties to the fusion proteinwhen used as a medicament. Surprisingly and most importantly, segment(b) allows production of said fusion protein in mammalian, preferablyhuman, cells and secretion to the cell supernatant in active form, i.e.in a form functional for binding to collagen. Segment (b) is mostpreferably an Fc domain of an immunoglobulin. Suitable immunoglobulinsare IgG, IgM, IgA, IgD, and IgE. IgG and IgA are preferred. IgGs aremost preferred. Said Fc domain may be a complete Fc domain or afunction-conservative variant thereof. A variant of Fc isfunction-conservative if it retains at least one of the functions ofsegment (b) listed above. Most preferred is the polypeptide of residues273 to 504 of SEQ ID No. 1. It is, however, general knowledge that sucha polypeptide may be modified or truncatated without abolishing itsfunction.

Segments (a) and (b) of the fusion protein of the invention may belinked by a linker. The linker may consist of about 1 to 100, preferably1 to 10 amino acid residues.

Most preferably, said fusion protein has the amino acid sequence of SEQID No. 1 (termed Fc-GPVI-nt herein).

The invention further provides a nucleic acid sequence coding for thefusion protein of the invention. Said nucleic acid sequence comprises asequence selected from the following group:

-   (i) the nucleic acid sequence of SEQ ID No: 2 or a variant thereof    that codes for the same polypeptide according to the degeneracy of    the genetic code;-   (ii) a nucleic acid sequence coding for a polypeptide that has at    least 70% sequence homology to the polypeptide encoded by SEQ ID No:    2;-   (iii) a nucleic acid coding for a polypeptide of at least 300 amino    acids, whereby a segment of at least 100 amino acids is functional    for binding to collagen and a segment of at least 200 amino acids is    functional as an Fc domain; and-   (iv) a nucleic acid sequence coding for the fusion protein of claim    1.

The invention further provides a medicament for the prevention ortreatment of intraarterial thrombosis, containing a protein thatcomprises the extracellular domain of glycoprotein VI or a variantthereof that is functional for binding to collagen. Preferably, saidprotein is said fusion protein of the invention. If said medicamentcontains said fusion protein, said medicament preferentially furthercomprises a suitable carrier. Said medicament is preferably administeredparenterally, more preferably it is administered intravenously. As hasbeen found by the present inventors, GP VI-collagen interactions are themajor factor of platelet adhesion to an injured vessel wall. The fusionprotein of the invention can prevent binding of platelets toblood-exposed collagen in the vascular system by blocking saidblood-exposed collagen without inhibiting other platelet functions.

Alternatively, the medicament of the invention may contain a nucleicacid that codes for said fusion protein of the invention for genetherepy. Said nucleic acid preferably contains the nucleic acid sequencedefined above. Said nucleic acid is preferably contained in a vector,preferentially a viral vector. Vectors encoding said fusion protein maybe introduced into the vascular system of a patient such that e.g.endothelial cells are transduced therewith. Suitable vectors for genetherapy are known in the art. They may be based e.g. on adenoviruses, onadeno-associated viruses, on retro viruses, or on herpes simplexviruses. Vectors may be adopted for long-term or for short-termexpression of the fusion protein by transduced cells, as the patientrequires. The Fc domain of the fusion protein enables secretion of thefusion protein in active form by transduced cells.

The invention further provides a method of in vitro screening forinhibitors of binding of glycoprotein VI to collagen, comprising

-   (i) providing a surface that exposes collagen;-   (ii) contacting a portion of said surface with the fusion protein of    the invention under predetermined conditions that allow binding of    said fusion protein to said surface;-   (iii) contacting another portion of said surface with said fusion    protein in the presence of a test compound under conditions as in    step (ii);-   (iv) determining the amount of said fusion protein bound to said    surface in the absence and in the presence of said test compound;-   (v) identifying a test compound as inhibitor if binding of said    fusion protein to said surface is less in the presence of said test    compound as compared to the absence of the test compound; and-   (vi) optionally determining the functional effect of said inhibitor    on platelet aggregation and/or platelet activation.

The surface of step (i) may be a glass or plastic surface coated withcollagen. The portions of said surface may be the wells of a titer plateor a multi-well plate. A surface that exposes collagen may be easilyprepared by coating a glass or plastic surface with collagen asdescribed in the examples. Collagen-coated plates or multi-well platesare also commercially available. In step (ii), a predetermined amount ofsaid fusion protein is contacted with a first portion of said surfaceunder conditions (notably pH, buffer, temperature) that allow binding ofthe fusion protein to the surface. Preferably, conditions are chosenthat allow optimal binding to said surface. In step (iii), anothersurface portion is contacted with the same amount of fusion protein andunder the same conditions as in step (ii) in the presence of apredetermined amount or concentration of a test compound. More than oneamount or concentration of a test compound may be used. Said determiningof step (iv) preferably comprises washing of said surface portionscontacted according to steps (ii) and (iiii) one or more times in orderto remove unbound fusion protein. The amount of bound fusion protein maythen be determined e.g. by measuring the fluorescence of a fluorescentlabel (e.g. fluorescein, rhodamine etc.) attached to the fusion protein.Alternatively, bound fusion protein may be detected using an antibodyagainst said fusion protein, whereby said antibody may be fluorescentlylabelled. Alternatively, the antibody may be labelled with an enzyme(e.g. alkaline phosphatase, a peroxidase, luciferase) capable ofproducing a coloured or luminescent reaction product. Most conveniently,the fusion protein may be labelled with a chromogenic label such thatthe label changes its light absorption or light emission characteristicsupon binding to collagen. In this embodiment, washing off of unboundfusion protein is not needed.

In step (v), inhibitors may identified. Identified inhibitors orselected moieties thereof may be used as lead structures for improvementof the inhibitor. Such lead structures may be modified using chemicalmethods and the modified structures may again be tested with thisscreening method. Modified structures or test compounds with improvedinhibition properties may be selected and optionally further varied bychemical methods. In this way, iterative improvement of an inhibitor maybe achieved. The inhibitors identified using the screening methods ofthe invention are valuable as potential drugs against thrombosis andarteriosclerosis.

In step (vi), the functional effect of said inhibitor on plateletaggregation and/or platelet activation may be determined according tomethods described below, e.g. by intravital fluorescence microscopy.

Said screening method may be carried out on small, medium, or largescale depending on the number of test compounds to be tested. If manytest compounds are to be tested (e.g. libraries of chemical compounds),the screening method preferably takes the form of a high-throughputscreening (HTS). For HTS, the amount of bound fusion protein ispreferably detected using fluorescently labelled fusion protein.

The above screening method may also be adopted for screening forantibodies that inhibit binding of GP VI to collagen, notably antibodiesagainst the extracellular domain of GP VI. Such an antibody screeningmay be combined with e.g. hybridoma technology of generating monoclonalantibodies or any other antibody generating technique, whereby thefusion protein of the invention is preferably used as antigen.Antibodies in hybridoma cell supernatants may be used as said testcompounds.

The invention further provides antibodies produced by using the fusionprotein of the invention as immunogen. Moreover, use of an antibodyagainst GPVI is provided for the preparation of a medicament for theprevention of platelet adhesion at exposed subendothelial matrixcollagens in active atherosclerotic lesions as the initial trigger foracute coronary or carotid syndrome. Such indications may be diagnosed asdescribed below. Preferably, the patient is further characterized bysuffering from unstable atherosclerotic plaque. Said medicament ispreferably administered parenterally. Preferably, said antibodies aremonoclonal antibodies. Such antibodies may e.g. be prepared using thefusion protein of the invention as immunogen.

Furthermore, the invention provides a method of in vitro screening foran inhibitor of GPVI mediated adhesion of platelets to activeintravascular lesions, said method comprising the steps of

-   (i) providing a surface exposing collagen;-   (ii) contacting the surface with platelets under predetermined    conditions allowing for an adhesion of the platelets to the    collagen;-   (iii) measuring the adhesion of platelets in the presence of a test    compound; and-   (iv) identifying the test compound as an inhibitor of GPVI when the    adhesion of platelets to collagen is less in the presence of the    test compound as compared to the absence of the test compound; and-   (v) optionally determining the functional effect of said inhibitor    on platelet aggregation and/or platelet activation.

Platelets to be used in this method may be isolated according to knownprocedures (cf. example 7). They may be isolated from blood of mammalslike mice, rats, rabbits, pigs etc. Preferably, they are isolated fromhumans. Said platelets may be labelled e.g. with a fluorescent dye likefluorescein. The adhesion of platelets to said surface may be measuredas described in the examples. The test compounds for this method may besmall organic molecules. Preferably, the test compounds for this methodsare inhibitors identified in the above method of screening forinhibitors of binding of GP VI to collagen. In this way, the number ofcompounds to be screened using platelets can be significantly reducedand the likelihood of finding inhibitors functional with platelets canbe increased.

Method of in vivo screening for an inhibitor of GPVI mediated adhesionof platelets to active intravascular lesions, said method comprising thesteps of

-   (i) providing an in vivo model for active intravascular lesions;-   (ii) measuring the adhesion of platelets to an active intravascular    lesion in the presence of a test compound, and-   (iii) identifying the test compound as an inhibitor of GPVI when the    adhesion of platelets to the active intravascular lesion is less in    the presence of the test compound as compared to the absence of the    test compound.

Said in vivo model may be a suitable mammal like a mouse, a rat, arabbit etc. Preferably, it is a mouse. Platelets that are preferablyfluorescently labelled are introduced into the model prior to measuringthe adhesion of platelets to an active intravascular lesion in thepresence and in the absence of a test compound. Said test compound haspreferably been identified as an inhibitor in one of the above in vitroscreening methods. Adhesion of platelets to an active intravascularlesion may be carried out by using in vivo fluorescence microscopy asdescribed in example 8.

The present invention also provides a use of a fusion protein comprising

-   (a) the extracellular domain of glycoprotein VI or a variant thereof    that is functional for binding to collagen and-   (b) the Fc domain of an immunoglobulin or a function-conservative    part thereof, for the manufacture of a medicament for the treatment    of diabetes.

The fusion protein used for the manufacture of a medicament for thetreatment of diabetes is preferably a dimeric fusion protein. In orderto provide for the possibility of dimerisation, a hinge region must bepresent between domains (a) and (b) of the fusion protein. The hingeregion is required for allowing suitable orientation of the polypeptidechains and formation of inter-chain disulfide bonds. Accordingly, thehinge region must have a sufficient length and contain cystein residues,preferably at least two cystein residues. Preferably, the fusion proteincomprises residued 1 to 267 of SEQ ID No:1. The fusion protein is usedfor the treatment of acute complications of diabetes or for thetreatment of chronic progression of atherosclerosis in diabeticpatients. Preferably, the fusion protein is Fc-GPVI-nt.

The present invention also provides a method for the preparation of afusion protein of the invention (Fc-GPVI-nt), which comprises thefollowing steps:

-   (a) collecting 2 days after infection the culture supernatant of    Hela cells infected with an adenovirus for Fc-GPVI-nt coding for an    amino acid sequence as shown in FIG. 7;-   (b) centrifuging (3800 g, 30 min, 4° C.) the supernatant of step    (a);-   (c) filtrating (0.45 μm) the supernatant of step (b);-   (d) precipitating the immunoadhesin by addition of 1 vol. ammonium    sulfate (761 g/l) and stirring overnight at 4° C.;-   (e) pelletizing the proteins by centrifugation (3000 g, 30 min, 4°    C.),-   (f) dissolving the pelletized proteins of step (e) in 0.1 Vol PBS    and dialysed in PBS overnight at 4° C.;-   (g) clarifying the protein solution by centrifugation (3000 g, 30    min, 4° C.);-   (h) loading the solution of step (g) on a protein A column (HiTrap™    protein A HP, Amersham Pharmacia Biotech AB, Uppsala, Sweden);-   (i) washing the column with binding buffer (20 mM sodium phoshate    buffer pH 7.0, 0.02% NaN₃) until OD₂₈₀<0.01;-   (k) eluting fractions with elution buffer (100 mM glycine pH 2.7);-   (l) neutralizing the eluted fractions with neutralisation buffer (1    M Tris/HCl pH 9.0, 0.02% NaN₃);-   (m) pooling the fractions;-   (n) diaiysing the pooled fractions in PBS overnight at 4° C.,-   (O) aliquoting the dialysed product and freezing at −20° C.

DESCRIPTION OF THE FIGURES

FIG. 1 Platelet adhesion and aggregation following vascular injury ofthe common carotid artery in C57BL6/J mice in vivo. (a) Scanningelectron micrographs of carotid arteries prior to (left panels) and 2hrs after (right panels) vascular injury. Endothelial denudation inducesplatelet adhesion and aggregation, resulting in the formation of aplatelet-rich (lower left) thrombus. (b) Platelet-endothelial cellinteractions 5 min after vascular injury were investigated by in vivofluorescence microscopy of the common carotid artery in situ (blackcolumns). Animals without vascular injury served as controls (opencolumns). The left and right panels summarize transient and firmplatelet adhesion, respectively, of eight experiments per group.Platelets were classified according to their interaction with theendothelial cell lining as described²⁴ and are given per mm² of vesselsurface. Mean±s.e.m., asterisk indicates significant difference comparedto control, P<0.05. (c) Platelet aggregation following vascular injurywas determined by fluorescence microscopy in vivo (black columns).Animals without vascular injury served as controls (open columns).Mean±s.e.m., n=8 each group, asterisk indicates significant differencecompared to wild type mice, P<0.05. The microphotographs (right) showrepresentative in vivo fluorescence microscopy images in control animals(upper panel) or following vascular injury (lower panel). White arrowsindicate adherent platelets.

FIG. 2 Inhibition of GPVI abrogates platelet adhesion and aggregationafter vascular injury. (a) Platelet adhesion following vascular injurywas determined by intravital videofluorescence microscopy. Fluorescentplatelets were preincubated with 50 μg/ml anti-GPVI (JAQ1) Fab fragmentsor control rat IgG. Platelets without mAb preincubation served ascontrol. The left and right panels summarize transient and firm plateletadhesion, respectively. Mean±s.e.m., n=8 each group, asterisk indicatessignificant difference compared to control, P<0.05. (b) illustrates thepercentage of platelets establishing irreversible adhesion after initialtethering/slow surface translocation is. (c) Platelet aggregationfollowing vascular injury in vivo. Aggregation of platelets preincubatedwith tyrodes, irrelevant rat IgG, or anti-GPVI Fab (JAQ1) was assessedby fluorescence microscopy as described. Mean±s.e.m., n=8 each group,asterisk indicates significant difference compared to control, P<0.05.(d) The photomicrographs show representative in vivo fluorescencemicroscopy images illustrating platelet adhesion in the absence orpresence of anti-GPVI Fab (JAQ1) or control IgG.

FIG. 3 Platelet adhesion following endothelial denudation inGPVI-deficient mice. (a) JAQ1-treated mice lack GPVI. Upper panels:Platelets from mice pretreated with irrelevant control IgG (left) oranti-GPVI (JAQ1) (right) were incubated with FITC-labeled JAQ1 andPE-labeled anti-mouse CD41 for 10 min at room temperature and directlyanalyzed on a FACScan™. A representative dot blot of 3 mice per group ispresented. Lower panel: Whole platelet lysates from three control IgG orJAQ1-treated mice were separated by SDS-PAGE under non-reducingconditions and immunoblotted with FITC-labeled JAQ1, followed byincubation with HRP-labeled rabbit-anti-FITC mAb. (b) Scanning electronmicrographs of carotid arteries 2 hrs after vascular injury in controlanimals (upper panels) or GPVI-depleted mice (lower panels). Endothelialdenudation induced platelet adhesion and platelet aggregation in controlanimals. In contrast, only very few platelets attached along the damagedvessel wall in GPVI-depleted mice. Subendothelial collagen fibers arevisible along the denuded area. (c) Platelet tethering and firm plateletadhesion, (d) transition from initial tethering to stable arrest(percentage of tethered platelets), and (e) platelet aggregationfollowing vascular injury of the carotid artery was determined inGPVI-deficient (JAQ1-pretreated mice) or control IgG-pretreated mice(for details see Materials and Methods). The panels summarize plateletadhesion (transient and firm) and platelet aggregation in eightexperiments per group. Mean±s.e.m., asterisk indicates significantdifference compared to control IgG, P<0.05. (f) The photomicrographsshow representative in vivo fluorescence microscopy images illustratingplatelet adhesion in GPVI-deficient (JAQ1) and control IgG-treated mice.

FIG. 4 Platelet adhesion to the surface of collagen coated glasscoverslips under physiological flow conditions was assessed ex vivo.Left panel: Platelets from mice pretreated with irrelevant control IgGimmunoadhesin (control) (left) or anti-GPVI immunoadhesin (Fc-GP VI-nt)(right) were investigated for adhesion under physiological flowconditions. The number of platelets was assessed by FACS counting of thewashed coverslips at the end of each experiment. Platelet tethering asthe first step of platelet adhesion was assessed after 30 seconds andfirm platelet adhesion after 5 min under flow conditions. (for detailssee Example 6). The panels summarize transient and firm plateletadhesion in eight experiments per group. Mean±s.e.m., asterisk indicatessignificant difference compared to control IgG, P<0.05.

FIG. 5 Interaction of Fc-GP VI-nt with collagen was monitored in anELISA based assay. Adhesion of the immunoadhesin Fc-GP VI-nt consistingof the extracellular domain of GP VI and the FC part of an IgG tocollagen coated plates with increasing concentrations of Fc-GP VI-nt(0.5 μg to 10 μg) was investigated. The binding is visualised with asecondary antibody labelled with peroxidase directed to the Fc part ofFc-GP VI-nt. Peroxidase is finally detected by ELISA. In thisrepresentative experiment binding of Fc-GP VI-nt to collagen wasmonitored with sufficient affinity, which reached saturation at μgconcentrations.

FIG. 6 Interaction of the Fc-GP VI-nt with collagen and the possibilityto screen for GP VI inhibitors was demonstrated with the inhibitory antimouse GP VI antibody JAQ 1. Adhesion of the immunoadhesin Fc-GP VI-nt (2μg/well) to collagen coated ELISA plates is shown to be specific: theempty immunoadhesin Fc-nt did not show any binding. Thus, this providesan ELISA based assay for the screening against GP VI inhibitors with theupscale potential to high-throughput capacities.

FIG. 7 Amino acid sequence of Fc-GPVI-nt: SEQ ID No: 1.

FIG. 8 DNA-Sequence of immunoadhesin Fc-GPVI-nt: SEQ ID No. 2. Bases 1to 807 encode the extracellular domain of GP VI. Bases 817 to 1515encode the Fc part of the IgG.

FIG. 9 Characterization of GPVI-Fc. (a) upper panel: Fc-GPVI-nt andcontrol Fc lacking the extracellular GPVI domain were used for SDS-PAGEunder reducing conditions. Coomassie blue stain (left) andimmunoblotting with peroxidase-conjugated goat anti-human Fc antibody(right) identified Fc-GPVI-nt with a molecular mass of ˜80 kDa. Middlepanel. Immunoblotting of Fc, Fc-GPVI-nt, or human platelets using theanti-GPVI monoclonal antibody 5C4. 5C4 detected both adenovirallyexpressed Fc-GPVI-nt fusion protein and platelet GPVI, but not thecontrol Fc. Lower panel: Molecular mass under reducing (right) andnon-reducing (left) conditions. While the molecular mass of Fc-GPVI-ntwas approximately 80 kDa under reducing conditions, the complete nt with˜160 kDa protein was identified under non-reducing conditions. (b-d)Characterization of Fc-GPVI-nt collagen interactions. (b) Binding assaysusing different concentrations of soluble Fc-GPVI-nt and immobilizedcollagen (10 μg/ml) were performed to define Fc-GPVI-nt-collageninteractions. Bound Fc-GPVI-nt was detected by anti-Fc mAb antibody(dilution 1:10.000) and is given relative to the binding observed at 10μg/ml Fc-GPVI-nt. Fc-GPVI-nt binds to collagen in a saturable manner.Mean±s.e.m., n=6 each Fc-GPVI-nt concentration, asterisk indicatessignificant difference compared to 0 μg/ml Fc-GPVI-nt, P<0.05. (c, leftpanel) shows binding of Fc-GPVI-nt (20 μg/ml) to various substrates.Binding of Fc-GPVI-nt to BSA (10 μg/ml) or vWF (10 μg/ml) is given aspercentage of GPVI-dimer-binding to immobilized collagen. Binding ofFc-GPVI-nt did not occur to BSA or vWF, supporting the specificity ofFc-GPVI-nt binding. Mean±s.e.m., asterisk indicates significantdifference compared to collagen, P<0.05. (c, right panel) illustratesbinding of Fc-GPVI-nt (20 μg/ml) or Fc (20 μg/ml) to immobilizedcollagen (10 μg/ml). Bound Fc-GPVI-nt or Fc was detected by anti-Fc mAbantibody (dilution 1:10,000) and is given relative to the bindingobserved with Fc-GPVI-nt. Only Fc-GPVI-nt, but not Fc or anti-Fc mAbbinds to immobilized collagen. Mean±s.e.m., n=8 each group, asteriskindicates significant difference compared to Fc-GPVI-nt binding, P<0.05.(d) Fc-GPVI-nt (20 μg/ml) was preincubated for 10 min with differentconcentrations of soluble collagen. After incubation the plates werewashed and Fc-GPVI-nt binding was detected by peroxidase-conjugated goatanti-human IgG antibody (dilution 1:10.000). Fc-GPVI-nt binding is givenrelative to the binding observed in the absence of soluble collagen.Soluble collagen inhibits GPVI-Fc-dimer-dimer binding to immobilizedcollagen in a dose-dependent manner. Mean±s.e.m., n=3 each collagenconcentration, asterisk indicates significant difference compared to 0mg/ml collagen, P<0.05. (e) The difference of the binding affinitybetween the monomeric form of the GPVI-Fc fusion portein and Fc-GPVI-ntwas assessed in direct comparison. The binding of the monomer and dimerwas assessed on collagen type 1 coated ELISA plates. Increasingconcentrations of the GPVI fusion proteins bond to collagen in asturable manner. Here a Linewaver Burke plot is demonstrated foraffinity assessment (e). The affinity of the monomeric GPVI fusionprotein was about 10 times lower compared to equimolar concentrations ofthe dimeric form Fc-GPVI-nt.

FIG. 10 Fc-GPVI-nt inhibits CD 62 P activation on human platelets as aparameter of release of intracellular transmitter substances from alphagranules by increasing doses of collagen. Human platelets were isolatedfrom whole blood and incubated with anti-CD 62 antibodies labelled withPE (for details see Material and Methods). Fluorescence was determinedin a Becton Dickenson FACS device. Representative histogramms are shown.Increasing concentrations of collagen from 0 to 10 μg/ml induced a shiftof fluorescence in the presence of the control Fc protein (100 μg/ml;blue line). In the presence of Fc-GPVI-nt (100 μg/ml; red line), theshift of fluorescence and hence CD 62 P activation was markedlyinhibited.

FIG. 11 Specific inhibition of collagen-mediated platelet aggregationand release of endogenous transmitters from dense and alpha granules byFc-GPVI-nt. (a) Human platelets were incubated with control Fc (80μg/ml) or Fc-GPVI-nt (80 μg/ml). Aggregation of platelets was inducedwith collagen (1 μg/ml) or ADP (5 μM) or TRAP (10 μM) and aggregationwas determined in an aggregometer under stirring conditions (for detailssee Material and Methods). Triplet measurements from n=5 different blooddonors were carried out. The means±s.e.m are given in % aggregation ofthe control aggregation without fusion proteins. (b) ATP release wasmeasured simultaneously in the same probes after incubation with controlFc (80 μg/ml) or Fc-GPVI-nt (80 μg/ml). The amount of ATP release isgiven in % of controls without fusion protein. (c) PDGF release wasdetermined in human platelets with an ELISA system specific for humanPDGF under basal conditions and after collagen (20 μg/ml) stimulation(for details see Material and Methods). Preincubation with control Fchad no significant effect on PDGF release from collagen-stimulatedplatelets, whereas Fc-GPVI-nt (100 μg/ml) reduced the PDGF releasesignificantly. Inhibition of PDGF release did not occur in unstimulatedplatelets.

FIG. 12 Fc-GPVI-nt has no significant effect on bleeding time in humanblood ex vivo. Bleeding time in human blood was measured ex vivo afterADP/collagen stimulation and epinephrine/collagen stimulation in aPFA-100 device. Fc-GPVI-nt (5 and 20 μg/ml) and Fc (5 and 20 μg/ml) didnot prolong bleeding time whereas ReoPro^(R) in a therapeuticallyrelevant concentration (5 μg/ml) maximally prolonged bleeding time underboth conditions. The means±s.e.m. from n=4 blood donors with tripletmeasurements are summarized.

FIG. 13 Fc-GPVI-nt inhibits platelet adhesion to immobilized collagenunder flow conditions. Human platelets (2×10⁸ cells/ml) were isolatedfrom whole blood (for details see “materials and methods”). Plates werecoated with immobilized collagen (10 μg/ml) or vWF (10 μg/ml). Plateletadhesion to the coated plates was determined in a parallel plate flowchamber in the presence of Fc-GPVI-nt or Fc lacking the extracellularGPVI domain (200 μg/ml). Inhibition of platelet adhesion by Fc-GPVI-ntis given in % of control (Fc control). Fc-GPVI-nt significantlyattenuated platelet adhesion on immobilized collagen at shear rates of500 sec⁻¹ and 1000 sec⁻¹, respectively. In contrast, Fc-GPVI-nt did notaffect platelet adhesion on immobilized vWF. Mean ±s.e.m., n=4 eachgroup, asterisk indicates significant difference compared to control Fc,P<0.05. The lower panels show representative microscopic images.

FIG. 14 Fc-GPVI-nt has favourable pharmacokinetics with a prolongedplasma half life after intraperitoneal injection in mice in vivo. Bloodconcentrations of Fc-GPVI-nt were determined with specific anti-Fcantibodies and ELISA (for details please see “material and methods”).(a) Single intraperitoneal injection of Fc-GPVI-nt (4 μg/g) led to rapidpeak blood concentrations of Fc-GPVI-nt after ˜24 h with slow decline ofFc-GPVI-nt blood concentrations. The means±s.e.m. from 10 animals aredemonstrated. (b) Repeated intraperitoneal applications (10 μg/g; twiceweekly) leads to continous accumulation of Fc-GPVI-nt in mice in vivoover 28 days. The means±s.e.m. from 6 animals are demonstrated. (c)Intravenous single dose injection of 30 μg Fc-GPVI-nt (1 μg/g bodyweight); 60 μg (2 μg/g body weight) and 100 μg Fc-GPVI-nt (3 μg/g bodyweight) per mouse led to a dose-dependent increase of immunoadhesinplasma concentration. The plasma concentration in the two higher dosesin these mice in vivo reached prolonged elevated levels from 5 to 60minutes and after 24 hours, sufficient for effective collagen scavengingand therefore effective inhibition of GPVI receptor activation onplatelets. The means±s.e.m. from 5 animals are demonstrated.

FIG. 15 Effects of Fc-GPVI-nt on platelet adhesion and aggregation invivo. (a) Mice (n=6 per group) were treated with 2 mg/kg or 4 mg/kgFc-GPVI-nt iv. Integrilin (0,2 mg/kg)-treated mice served as positivecontrols (n=8). Bleeding times were determined as described (see“materials and methods”). The Fc-GPVI-nt fusion protein did not increasetail bleeding times compared to control animals. In Integrilin-treatedmice tail bleeding time was massively prolonged. **P<0.05 vs. control.(b) Inhibition of GPVI abrogates platelet adhesion and aggregation aftervascular injury. Platelet adhesion following vascular injury wasdetermined by intravital video fluorescence microscopy. Mice werepretreated with 1 or 2 mg/kg Fc-GPVI-nt or equimolar amounts of controlFc. The left and right panels summarize platelet tethering and firmplatelet adhesion, respectively. Mean±s.e.m., n=5 each group, asteriskindicates significant difference compared to Fc, P<0.05. (c) Effects ofFc-GPVI-nt on thrombus formation following vascular injury in vivo. Thenumber of platelet thrombi (right) and the total thrombus area (left)were assessed by fluorescence microscopy as described. Mean±s.e.m., n=5each group, asterisk indicates significant difference compared to Fc,P<0.05. (d) The photomicrographs show representative in vivofluorescence microscopy images illustrating platelet adhesion in theabsence or presence of 1 or 2 mg/kg Fc-GPVI-nt or control Fc. Barsrepresent 501 μm. (e) Scanning electron micrographs of carotid arteries1 hr after vascular injury in Fc- or Fc-GPVI-nt treated animals.Endothelial denudation induced platelet adhesion and plateletaggregation in Fc-treated mice. In contrast, only very few plateletsattached along the damaged vessel wall in Fc-GPVI-nt-treated mice.Subendothelial collagen fibers are visible along the denuded area. Barsrepresent 10 μm (f) Fc-GPVI-nt specifically binds to the subendotheliumof carotid arteries. The binding of Fc-GPVI-nt to the subendothelium wasdetermined on carotid sections, stained with peroxidase-conjugated goatanti-human IgG antibody. Carotid arteries obtained from Fc-treated miceserved as controls. Fc-GPVI-nt but not Fc control protein was detectedat the subenothelium, as indicated by the brown staining. Originalmagnification: 100-fold.

FIG. 16 Fc-GPVI-nt significantly attenuates atheroprogression in apo e−/− knockout mice in vivo. Apo e −/− mice were treated with Fc-GPVI-nt(4 μg/g) or control Fc (4 μg/g) intraperitoneally for 4 weeks twiceweekly. Atheroprogression was investigated post mortem after sudan redstaining of the large vessels to visualise atheroma and plaqueformation. In control animals extensive plaque formation of carotidartery preparations was indicated by the red colour in particular in thebranching region. In Fc-GPVI-nt treated animals atherosclerosis wasalmost completely abolished in carotid arteries of apo e −/− mice.Representative macroscopic whole vascular preparations of the carotidearteries of an apo e −/− mouse after 4 weeks treatment with Fc-GPVI-nt(left side) and of an apo e −/− mouse after 4 weeks treatment with thecontrol Fc protein (right side) are shown.

FIG. 17 Freshly isolated platelets from patients suffering from diabetesmellitus show reduced expression of the fibrinogen receptor (CD61, top)and increased expression of the Fc receptor (CD32, middle) and thereforeincreased expression of GPVI. The correleation between CD32 expressionand GPVI expression (detected by the specific monoclonal antibody 4C9)is shown on human platelets (bottom). Human platelets were isolated fromwhole blood from patients suffering from diabetes and incubated withfluorescent anti-CD61 and anti CD32 antibodies or FITC labelled 4C9antibodies. Fluorescence was determined in a Becton DickensonFACScalibur device. The means+/−s.e.m. from n=111 diabetic patients andfrom n=363 patients without diabetes are summarized. Correlation of CD32fluorescence and 4C9 fluorescence was calculated with the correlationcoefficient r=0.516.

FIG. 18 Amino acid sequence of a monomeric fusion protein based onFc-GPVI-nt.

DETAILED DESCRIPTION OF THE INVENTION

A previous hypothesis suggested that platelet glycoprotein (GP) lbbinding to von vWf recruits flowing platelets to the injured vessel wall(Ruggeri, Z. M:. Mechanisms initiating platelet thrombus formation.Thromb. Haemost 1997; 78, 611-616), whereas subendothelial fibrillarcollagens support firm adhesion and activation of platelets (van Zanten,G. H. et al. Increased platelet deposition on atherosclerotic coronaryarteries. J. Clin. Invest 1994; 93, 615-632; Clemetson, K. J. &Clemetson, J. M. Platelet collagen receptors. Thromb. Haemost. 2001, 86,189-197). However, the present invention demonstrates by in vivofluorescence microscopy of the mouse carotid artery that inhibition orabsence of the major platelet collagen receptor, GPVI, instead,abolishes platelet-vessel wall interactions following an endothelialerosion. Unexpectedly, inhibition of GPVI reduces platelet tethering andadhesion to the subendothelium by approximately 89%. Furthermore, stablearrest and aggregation of platelets is virtually abolished under theseconditions. The strict requirement for GPVI in these processes wasconfirmed in GPVI-deficient mice, where platelets also fail to adhereand aggregate on the damaged vessel wall. These findings reveal anunexpected role of GPVI in the initiation of platelet attachment atsites of vascular injury and unequivocally identify platelet-collageninteractions as the major determinant of arterial platelet-inducedatherosclerotic complications.

The fact that GP VI generally functions as a receptor for thesubendothelial matrix collagen has been described (Moroi M, Jung S M,Okuma M, Shinmyozu K. A patient with platelets deficient in glycoproteinVI that lack both collagen-induced aggregation and adhesion. J ClinInvest 1989; 84: 1440-1445). These authors characterized platelets invitro originating from patients with a GP VI receptor deficiency.However, the physiological significance of the interaction of collagenand GP VI receptor in the in vivo context and the relative contributionof the GP VI receptor for adhesion following vascular injury wasunknown. In particular, it was not known that inhibition of thisreceptor inhibits the key step in the formation of intravascularthrombosis that is platelet tethering. The present invention reveals theGP VI receptor as an essential receptor for platelet adhesion to thesubendothelium via the attachment to subendothelial matrix collagen invivo. Amongst the variety of other platelet surface proteins such as GPlb (von Willebrand receptor), the αIIbβ3 integrin receptor, the α2β1integrin or the GP V receptors, we have surprisingly identified the GPVI receptor to be an essential receptor to mediate platelet adhesion tothe vascular wall. Since platelet adhesion is the first and mostimportant step for platelet aggregation and intraarterial thrombusformation under physiologic shear stress conditions, the followingdeleterious effects leading to intraarterial occlusion are thefunctional basis for the clinical syndromes of myocardial infarction orcerebral stroke. In a chronic setting, the interaction of platelets withthe endothelium propagates early steps of arteriosclerosis. Ourinvention also showed for the first time that the GP VI receptor plays acrucial role amongst the complex variety of several platelet surfaceproteins for initial platelet adhesion and for chronicalplatelet—endothelium interaction in the propagation of arteriosclerosis.

WO 01/16321 and WO 01/00810 disclose a DNA and protein sequence of thehuman GPVI receptor. However, the significance on platelet adhesion andactivation by endothelial lesions has not been demonstrated in an invivo background.

U.S. Pat. No. 6,383,779 discloses fusion proteins of GPVI. However, thisreference does not disclose a dimeric fusion protein or any therapeuticeffect of GPVI.

Recently, the different phases of platelet-collagen interaction toartificial collagen in vitro during perfusion conditions wereinvestigated (Moroi M, Jung S M, Shinmyozu K, Tzomiyama Y. Ordinas A andDiaz-Ricart M. Analysis of platelet adhesion to collagen-coated surfaceunder flow conditions: the involvement of glycoprotein VI in theplatelet adhesion. Blood 1997; 88: 2081-2092). The authors of that studyalready pointed out the importance of collagen-GP VI interaction duringshear stress conditions. However, the relevance of subendothelial matrixcollagen for the adhesion could not be studied in this artificial invitro situation. As a consequence of limited relevance of their in vitromodel, the authors of the above mentioned study came to the conclusionthat GP VI receptors are rather involved in platelet activation than inplatelet adhesion to the endothelium. In contrast, the von Willebrand GPlb receptor is significantly involved in platelet-subendothelialinteraction. These authors also focussed all available information aboutplatelet—collagen interaction in a review of the current literature.Previously, Moroi M and Jung, S M (Platelet receptors for collagen.Thromb. Haemost. 1997; 78: 439-444) have discussed collagen fibrilinteraction with different collagen receptors on platelets for theadhesion and thrombus formation of platelets. However, the authors didnot expect a relevant role of the GP VI receptor for the adhesion in aclinically relevant in vivo situation as they could not validate thesignificance of the different collagen receptors to the adhesionprocess.

Therefore, the present invention provides a solution to the problem ofinhibiting the relevant target for the platelet—subendothelialinteraction and for platelet adhesion without provoking undesired sideeffects of bleeding complications. Besides the well known interaction ofcollagen —platelet via the GP VI receptor, we could provide data for theinteraction of the native subendothelial matrix and platelets measuredby in vivo platelet adhesion. Consecutively, we could validate thesignificance of the GP VI-endothelium interaction for platelet adhesionas initial step of intravascular thrombosis. Thus, our invention solvesthe problem of an effective antiplatelet drug treatment for theimportant step of platelet adhesion without undesired side effects.

Further, the invention provides an immunoadhesin (the fusion protein ofthe invention). In a specific embodiment, the immunoadhesin consists ofthe extracellular domain of the GP VI receptor together with the Fc partof an IgG immunoglobulin (Fc-GPVI-nt). This novel fusion protein isbased approximately 50% on the original DNA sequence of GP VI aspublished previously. The protein structure of the immunoadhesin isnovel as the recombinant fusion protein does not form a membrane proteinlike the GP VI receptor but is a soluble, immunoglobulin-likeimmunoadhesin released by the respective host cell. This immunoadhesincan block the ligand-receptor interaction of collagen and GP VI. Ourresults demonstrate that the immunoadhesin has marked effects on themain physiological functions of platelets induced by collagenstimulation. Collagen-induced aggregation, adhesion and the releasefunction can be inhibited by the immunoadhesin to the same extent asdoes a specific, monoclonal antibody. The mechanism, however, isdifferent: whereas the antibody inhibits GP VI activation by directlybinding to the ligand binding site of the GP VI receptor, theimmunoadhesin scavenges the GP VI ligand collagen and therefore preventsligand-mediated GP VI activation.

The immunoadhesin of the invention is a novel GP VI inhibitor. It hasthe advantage of selective inhibition of the activated branch of GP VImediated effects by ligand scavenging. Secondary effects, like antibodymediated effects on GP VI receptor internalisation are prevented.Fc-GPVI-nt can be used for the treatment of atheroscleroticcomplications caused by unstable atheroslerotic plaques with plaquerupture or endothelial lesion. Therefore, the immunoadhesin Fc-GPVI-ntserves as a therapeutic inhibitor for collagen-mediated GP VI activationwithout affecting the intrinsic activity of the GP VI receptor with therelevant signalling system.

Moreover, the GP VI immunoadhesin serves as an ideal epitope forantibody selection. The Fc part allows the convenient purification ofthe protein and simple fixation to surfaces to perform large scaleantibody selection against antibody libraries i.e. by phage display. Theselection allows selective antibody screening to the relevant epitopethat resembles the intact protein with a similar structure as the nativeprotein.

Finally, the Fc-GPVI-nt is an important tool for the screening forinhibitors of GP VI receptor activation. We have established anELISA-based in vitro assay simulating the collagen GP VI interaction bycollagen precoated plates as the ligand. This assay can alternatively berun with fluorescence-labelled Fc-GPVI-nt and thus be upscaled tohigh-throughput formats. This assay allows for the screening of both,inhibitory antibodies or small molecules for their potency to inhibit GPVI function by fluorescence measurement With this cell free screeningassay, a prototype method for a high-throughput-scaleable fluorescencescreening assays for drug testing has been established.

Based on the recent improvements in imaging techniques by intravascularultrasound or nuclear magnetic resonance imaging, it is possible toidentify patients with atherosclerosis being at risk of acute clinicalcomplications such as acute coronary or carotid syndrome, whereby thepatients have active lesions as possible causes for intravascularthrombosis. It is then possible by the present invention to prevent theformation of intravascular thrombosis by the administration of amedicament containing an antibody against platelet glycoprotein VI(GPVI) without undesired side effects.

Active lesions are characterized by the unmasking of subendothelialmatrix collagens and platelet activation. The occurrence of such lesionscan be investigated e.g. by intravascular ultrasound or thermography(e.g., Fayed and Fuster, Clinical imaging of the high-risk or vulnerableatherosclerotic plaque. Circulation 2001; 89:305-316) or nuclearresonance imaging (Helft et al., Progression and Regression ofAtherosclerotic Lesions. Circulation 2002; 105:993-998). Such lesionsare highly probable in patients with acute coronary or carotidsyndromes, and the risk of the reoccurrence of acute clinicalcomplications such as myocardial infarction or stroke is very high,decreasing progressively with increasing time distance from the primaryevent.

Therefore, the present invention also provides a method of treating apatient suffering from an acute coronary or carotid syndrome, saidmethod comprising for avoiding intravascular thrombosis the steps of

-   (a) determining the presence or absence of active intravascular    lesions in the patient; and-   (b) treating the patient with an antibody against platelet    glycoprotein VI (GPVI) in case of the presence of intravascular    lesions.

Moreover, based on the present invention, it is possible to treatpatients being at risk of intravascular thrombosis due to the rupture ofcomplex arteriosclerotic plaques. The rupture also unmasks thesubendothelial collagen matrix. As a consequence of intraarterialthrombus formation, the perfusion of vital organs is blocked with theabove described important and life threatenting clinical syndromes.

The present invention also provides a method of treating a patientsuffering from a chronic atherosclerotic syndrome, said methodcomprising for avoiding intravascular thrombosis the steps of

-   (a) determining the presence or absence of the onset of    atheroprogression in the patient; and-   (b) treating the patient with an antibody against platelet    glycoprotein VI (GPVI) in case of the presence of intravascular    lesions.

Accordingly, based on the present invention, it is possible to treatpatients being at risk of atherosclerosis. In order to preventatheroprogression, a patient is treated with the fusion protein of theinvention in order to prevent interaction between platelets and exposedsubendothelial collagen. The fusion protein of the invention blocks theligand for the GPVI platelet receptor in the vascular wall (e.g.subendothelium) so that an interaction between the platelets and exposedcollagen is inhibited.

The fusion protein of the invention may be in the form of a lyophilisedpowder which is dispersed in a suitable pharmaceutically acceptableliquid carrier prior to administration to a patient. The fusion proteinof the invention can also be incorporated into pharmaceuticalcompositions suitable for parenteral, in case of the treatment of acutecomplications preferably intraarterial or intravenous administration.Such compositions usually comprise the fusion protein and apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier includes solvents, dispersion media, antibacterial andantifungal agents and isotonic agents, which are compatible withpharmaceutical administration. The present invention includes methodsfor manufacturing pharmaceutical compositions for the treatment ofchronic or acute cardiovascular disease. Such methods compriseformulating a pharmaceutically acceptable carrier with the fusionprotein of the invention. In case of the treatment of acutecardiovascuar disease, the composition is preferably administeredintravenously or intraarterially. In case of the treatment of chroniccardiovascular disease, the composition may also be administeredsubcutaneously and intraperitoneally. Such compositions can furtherinclude additional active compounds, such as further polypeptides (suchas insulin) or therapeutically active small molecules. Thus, theinvention further includes methods for preparing a pharmaceuticalcomposition by formulating a pharmaceutically acceptable carrier withthe fusion protein of the invention and one or more additional activecompounds such as insulin. In case of the coformulation of the fusionprotein and insulin for the treatment of diabetic patients, it ispreferred that the dosage form allows separate storage of the differentproteins whereby mixing of the proteins is carried out just prior orduring the administration of the composition. Accordingly, applicationby a multi-chamber syringe is considered. A pharmaceutical compositionof the invention is formulated to be compatible with its intendedparenteral route of administration. Examples of routes of parenteraladministration include, e.g., intraarterial and intravenousadministration. Solutions or suspensions used for parenteral may includea sterile diluent such as water for injection, saline solution,polyethylene glycols, fixed oils, glycerine, propylene glycol, TWEEN orother synthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; chelating agents such as ethylenediaminetetraaceticacid; antioxidants such as ascorbic acid or sodium bisulfite; bufferssuch as acetates, citrates or phosphates and agents for the adjustmentof tonicity such as sodium chloride, dextrose, saccarose or mannitose.The pH can be adjusted with acids or bases, such as hydrochloric acid orsodium hydroxide. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, or phosphate buffered saline(PBS). The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol), and suitable mixtures thereof.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of micoorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin. Sterile injectable solutionscan be prepared by incorporating the active compound (e.g., apolypeptide or antibody) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. It is especiallyadvantageous to formulate oral or parenteral compositions in dosage unitform. A dosage unit form are discrete units suited as unitary dosagesfor a patient. Each unit contains a predetermined quantity of activecompound to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. A therapeutically effective amountof fusion protein (i.e., an effective dosage) for the treatment of acutecomplications ranges from 0.05 to 5 mg/kg body weight, preferably 0.1 to2 mg/kg body weight, more preferably 0.1 to 1 mg/kg body weight. Atherapeutically effective amount of fusion protein (i.e., an effectivedosage) for the treatment of chronic atheroprogression ranges from 0.5to 6 mg/kg body weight, preferably 1 to 5 mg/kg body weight, morepreferably 2 to 5 mg/kg body weight. The treatment of a subject with atherapeutically effective amount of the fusion protein can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with the fusion protein of theinvention against chronic atheroprogression in the range of between 0.5to 6 mg/kg body weight, preferably 1 to 5 mg/kg body weight, morepreferably 2 to 5 mg/kg body weight, at least twice per week.

Methods to Investigate Platelet-Collagen Interaction and Modulation byInhibitors

Platelet Aggregation and ATP Release

Stimulation of mouse platelet-rich plasma with increasing concentrationsof bovine type I collagen from 0.2 to 4 μg/ml elicits a dose-dependentaggregation from 2 to 95% and a dose-dependent ATP release from 0 to1.66 nM ATP release. A half-maximal collagen concentration was chosenfor further experiments. Incubation of the mouse platelet-rich plasmawith the specific anti-mouse GP VI antibody JAQ 1 (50 μg/ml and 100μg/ml) almost completely abolished platelet aggregation afterstimulation with 2 μg collagen/ml (with 50 μg JAQ 1:2+/−0.7; with 100 μgJAQ 1:1.5+/−0.3%). Moreover, ATP release was inhibited in an antibodydose-dependent manner to 1.09 nM ATP (10 μg antibody/ml) or completelyabolished (50 and 100 μg antibody/ml).

Similarly, incubation of mouse platelet-rich plasma with theimmunoadhesin for GP VI (Fc-GPVI-nt) (50 μg/ml and 100 μg/ml) almostcompletely abolished platelet aggregation after stimulation with 2 μgcollagen/ml (with 50 μg Fc-GPVI-nt: 2+/0.7; with 100 μg Fc-GPVI-nt 1.5+/−0.3%) and ATP release to 0 nM ATP.

Therefore, the immunoadhesin sufficiently inhibited GP VI activation byscavenging the natural GP VI ligand collagen. Both the crucial plateletfunction aggregation and the platelet release mechanism as determined byATP release could be influenced by the Fc-GPVI-nt.

GP VI Mediated Adhesion Under Physiological Flow Conditions (FlowChamber)

Adhesion of platelets under physiological shear conditions was tested ina flow chamber. Initial and firm adhesion of platelets was significantlyinhibited by addition of the Fc-GPVI-nt immunoadhesin by 60% (see FIG.4).

GP VI Binding Assay

Adhesion of Fc-GPVI-nt to collagen coated plates was determined in anELISA based fluorescence assay. The binding of the immunoadhesinFc-GPVI-nt dose dependently increased up to saturation levels in aconcentration from 0.2 to 10 μg Fc-GPVI-nt (please see FIG. 5). Thespecificity was demonstrated by comparing binding of Fc-GPVI-nt withthat of the empty immunoadhesin Fc-nt or the uncoated plastic surface(see FIG. 6).

METHODS to Investigate Platelet Adhesion and Aggregation at VascularInjury In Vivo as the Crucial Steps for Platelet Activation in AcuteVascular Events

To test the biological significance of platelet-collagen interactions inthe processes of adhesion to lesions in vivo, platelet-vessel wallinteractions following vascular injury of the mouse carotid artery areassessed. Vascular injury to this important vascular bed may serve as amodel for the first steps of arteriosclerosis such as the endotheliallesion in early stage arteriosclerosis or the plaque rupture in laterstages of arteriosclerosis with the unmasking of collagen fibrils fromthe subendothelium. Moreover, this model allows the study of thesubsequent complications of vascular injury. Small endothelial lesionslead to maximal activation of platelets with the following steps ofplatelet adhesion and aggregation. In further steps platelet aggregatescan lead to embolism from the carotid artery with consecutive ischemiccerebral stroke. Thus, this experimental setup serves as a relevant invivo model for a subgroup of patients with unstable atherosclerosisinvolving plaque rupture and endothelial lesions leading to acutecoronary syndrome and stroke.

Vigorous ligation of the carotid artery for 5 min consistently causescomplete loss of the endothelial cell layer and initiates plateletadhesion at the site of injury, as assessed by scanning electronmicroscopy (FIG. 1 a). In vivo fluorescence microscopy may be used todirectly visualize and quantify the dynamic process of plateletaccumulation following vascular injury. Numerous platelets are tetheredto the vascular wall within the first minutes after endothelialdenudation (4620±205 platelets/mm²). Virtually all plateletsestablishing contact with the subendothelium exhibit initially a slowsurface translocation of the “stop-start” type (Savage, B., Saldivar, E.& Ruggeri, Z. M. Initiation of platelet adhesion by arrest ontofibrinogen or translocation on von Willebrand factor. Cell 1996; 84,289-297). While we observed transition from initial slow surfacetranslocation to irreversible platelet adhesion in 88% of all platelets(4182±253 platelets/mm²) (FIG. 1 b), platelet arrest remains transientin only 12% (543±32 platelets/mm²). Once firm arrest is established,adherent platelets recruit additional platelets from the circulation,resulting in aggregate formation (FIG. 1 c). Similar characteristics ofplatelet recruitment are obtained with immobilized collagen in vitro. Incontrast, only few platelets are tethered to the intact vascular wallunder physiological conditions (P<0.05 vs. vascular injury) andvirtually 100% of these platelets are displaced from the vascular wallwithout firm arrest (P <0.05 vs. vascular injury, FIG. 1 a-c).

Identification of GP VI as a Novel and Relevant Target Protein inPlatelets for Vascular Injury in Vivo

The high complexity of the platelet-vessel wall interaction whichinvolves a variety of different receptors and signaling pathways makesthe in vivo inhibition of this process very difficult. BesidesGPIb-V-I-X and α_(IIb)β₃ integrin which interact indirectly withcollagen via von Willebrand factor (vWF), a large number of collagenreceptors have been identified on platelets, including most importantlyα₂β₁ integrin (Santoro, S. A. Identification of a 160,000 daltonplatelet membrane protein that mediates the initial divalentcation-dependent adhesion of platelets to collagen. Cell 1986; 46,913-920), GPV (Moog, S. et al. Platelet glycoprotein V binds to collagenand participates in platelet adhesion and aggregation. Blood 2001; 98,1038-1046), and GPVI (Moroi, M., Jung, S. M., Okuma, M. & Shinmyozu, K.A patient with platelets deficient in glycoprotein VI that lack bothcollagen-induced aggregation and adhesion. J. Clin. Invest 84,1440-1445). Amidst several reports on different signaling systems whichplay a role in vitro, also GPVI has now been discussed (Gibbins, J. M.,Okuma, M., Farndale, R., Baames, M. & Watson, S. P. Glycoprotein VI isthe collagen receptor in platelets which underlies tyrosinephosphorylation of the Fc receptor gamma-chain. FEBS Lett. 1997; 413,255-259; Nieswandt, B. et al., Long-term antithrombotic protection by invivo depletion of platelet glycoprotein VI in mice. J. Exp. Med. 2001;193, 459469, Nieswandt, B. et al Glycoprotein VI but not α2β1 integrinis essential for platelet interaction with collagen. EMBO J. 2001; 20,2120-2130).

To directly test the in vivo relevance of platelet-collagen interactionsin arterial thrombus formation, we inhibited or deleted GPVI in vivo.The monoclonal antibody (mAb) JAQ1 blocks the major collagen-bindingsite on mouse GPVI (Schulte, V. et al. Evidence for two distinctepitopes within collagen for activation of murine platelets. J. Biol.Chem. 2001; 276, 364-368) and almost completely inhibits firm plateletadhesion to immobilized fibrillar collagen under high shear flowconditions (Nieswandt, B. et a. Glycoprotein VI but not alpha2beta1integrin is essential for platelet interaction with collagen. EMBO J.2001; 20, 2120-2130). To study the significance of GPVI-collageninteractions in the dynamic process of platelet adhesion/aggregation inarterial thrombus formation, mice received syngeneic,fluorescence-tagged platelets pre-incubated with JAQ1 Fab fragments orisotype-matched control IgG and carotid injury was induced as describedabove. Very unexpectedly, we found that the inhibition of GPVI reducedinitial platelet tethering following endothelial denudation in thecommon carotid artery by 89% (P<0.05 vs. control IgG, FIG. 2 a), aprocess thought to be mediated mainly by GPIbα interaction withimmobilized vWF (Goto, S., Ikeda, Y., Saldivar, E. & Ruggeri, Z. M.Distinct mechanisms of platelet aggregation as a consequence ofdifferent shearing flow conditions. J. Clin. Invest. 1998; 101, 479-486;Sixma, J. J., van Zanten, G. H., Banga, J. D., Nieuwenhuls, H. K. & deGroot, P. G. Platelet adhesion. Semin. Hematol. 1995; 32, 89-98).Furthermore, stable platelet arrest was reduced by 93% by JAQ1 (FIG. 2a). We observed transition from initial tethering/slow surfacetranslocation to irreversible platelet adhesion in only 58% of thoseplatelets establishing initial contact with the subendothelial surface(compared to 89% with control IgG-pretreated platelets, P<0.05, FIG. 2b). Aggregation of adherent platelets was virtually absent followingpretreatment of platelets with JAQ1 Fab fragments, but not in thecontrols (P<0.05 vs. control, FIGS. 2 c and d). These data demonstratedthat direct platelet-collagen interactions are crucial for initialplatelet tethering and subsequent stable platelet adhesion andaggregation at sites of vascular injury. Furthermore, these findingsshow that GPVI is a key regulator in this process, while other surfacereceptors, most importantly GPIb-V-IX and α₂β₁, are not sufficient toinitiate platelet adhesion and aggregation on the subendothelium invivo.

To exclude the possibility that this effect is based on stericimpairment of other receptors, e.g. GPIb-V-IX, by surface-bound JAQ1, wegenerated GPVI-deficient mice by injection of JAQ1 five days prior tovascular injury. As reported previously, such treatment inducesvirtually complete loss of GP VI e.g. by internalization and proteolyticdegradation of GPVI in circulating platelets, resulting in a “GPVI knockout”-like phenotype for at least two weeks (Nieswandt, B. et a.Long-term antithrombotic protection by in vivo depletion of plateletglycoprotein VI in mice. J. Exp. Med. 2001; 193, 459469). As illustratedin FIG. 3 a, GPVI was undetectable in platelets from JAQ1-treated miceon day 5 after injection of 100 μg/mouse JAQ1, but not control IgG,while surface expression and function of all other tested receptors,including GPIb-V-IX, α_(IIb)β3, and α₂β₁ was unchanged in both groups ofmice, confirming earlier results (data not shown and Nieswandt, B. etal., Long-term antithrombotic protection by in vivo depletion ofplatelet glycoprotein VI in mice. J. Exp. Med. 2001; 193, 459-469).

As shown by scanning electron microscopy, platelet adhesion andaggregation following endothelial denudation of the common carotidartery is virtually absent in GPVI-deficient, but not in IgG-pretreatedmice (FIG. 3 b). Next, in vivo video fluorescence microscopy was used todefine platelet adhesion dynamics following vascular injury inGPVI-deficient mice (FIG. 3 c-f). The loss of GPVI significantly reducestethering/slow surface translocation of platelets at the site ofvascular injury (by 83% compared to IgG-pretreated mice, P<0.05). ThisGPVI-independent slow surface translocation requiresvWF-GPIbα-interaction, since it is abrogated by preincubation of theplatelets with Fab fragments of a function blocking mAb againstGPIbα(p0p/B) confirming the critical role of GPIbα in this process (notshown). In the absence of GPVI, stable platelet adhesion is reduced byapproximately 90% compared to the (IgG-treated) control, whileaggregation of adherent platelets is virtually absent (FIG. 3 b-f). Wesaw transition from platelet tethering to stable platelet adhesion inonly 58% of all platelets initially tethered to the site of injury(compared to 89% with control mAb-pretreated platelets, P<0.05, FIG. 3d), indicating that GPIbα-dependent surface translocation is notsufficient to promote stable platelet adhesion and subsequentaggregation.

The profound inhibition of platelet tethering by GPVI blockade wassurprising and suggested a previously unrecognized function of thisreceptor in the very Initial phase of firm platelet adhesion to vascularlesions. Fibrillar collagen is a major constituent of humanatherosclerotic lesions (Rekhter, M. D. Collagen synthesis inatherosclerosis: too much and not enough. Cardiovasc. Res. 1999; 41,376-384; Rekhter, M. D. et al. Type I collagen gene expression in humanatherosclerosis. Localization to specific plaque regions. Am. J. Pathol.1993; 143, 1634-1648); enhanced collagen synthesis (by intimal smoothmuscle cells and fibroblasts) significantly contributes to luminalnarrowing in the process of atherogenesis (Opsahl, W. P., DeLuca, D. J.& Ehrhart, L. A. Accelerated rates of collagen synthesis inatherosclerotic arteries quantified in vivo. Arteriosclerosis 1987; 7,470-476). Plaque rupture or fissuring (either spontaneously or followingballoon angioplasty) results in exposure of collagen fibrils to theflowing blood.

The invention teaches for the first time that such subendothelialcollagens are the major trigger of arterial thrombus formation andreveal an unexpected function of the collagen receptor GPVI in plateletrecruitment to the injured vessel wall. The processes of platelettethering and slow surface translocation under conditions of elevatedshear are known to largely depend on GPIbα interaction with immobilizedvWF. This interaction is, however, not sufficient to establish initalplatelet-vessel wall interactions in vivo as functional GPVI is alsorequired (FIGS. 2 and 3). Thus, both GPIbα and GPVI must act in concertto recruit platelets to the subendothelium. During platelet tethering,ligation of GPVI can shift α_(IIb)β₃ and α₂β₁ integrins from a low to ahigh affinity state. Both α_(IIb)β₃ and α₂ β_(l) then act in concert topromote subsequent stable arrest of platelets on collagen, whileα_(IIb)β₃ is essential for subsequent aggregation of adherent platelets.Thus, ligation of GPVI during the initial contact between platelets andsubendothelial collagen provides an activation signal that is essentialfor subsequent stable platelet adhesion and aggregation. Importantly,occupation or lateral clustering of GPIbα (during GPIbα-dependentsurface translocation), which induced low levels of α_(IIb)β₃ integrinactivation in vitro (Kasirer-Friede, A. et al. Lateral clustering ofplatelet GP Ib-IX complexes leads to up-regulation of the adhesivefunction of Integrin αIIbβ3. J. Biol. Chem. 2002; Vol 277: 11949-11956),is not sufficient to promote platelet adhesion in vivo.

The invention therefore has identified an essential receptor forinhibiting platelet attachment to the subendothelium. An antibody whichblocks the interaction of GPVI with exposed collagen can specificallyinhibit all major phases of thrombus formation, i.e. platelet tethering,firm adhesion, and aggregation at sites of arterial injury (e.g. duringacute coronary syndromes). The very profound protection that wasachieved by inhibition or depletion of GPVI establishes the importanceof selective pharmacological modulation of GPVI-collagen interactions tocontrol the onset and progression of pathological atheroscleroticlesions.

Following rupture of the atherosclerotic plaque, exposure ofsubendothelial collagen is the major trigger that initiates plateletadhesion and aggregation at the site of injury, followed by arterialthrombosis (1;24;25). The platelet glycoprotein GPVI, which has beencloned recently (5;6), has been identified by the invention to be themajor platelet collagen receptor (4), mediating platelet adhesion bothin vitro (22) and under (patho-)physiological conditions in vivo (3).Therefore, inhibition of GPVI prevents platelet recruitment and arterialthrombosis in patients with advanced atherosclerosis as shown by thepresent invention by the inhibitory activities of the specific fusionprotein Fc-GPVI-nt on platelet adhesion in vitro and in vivo.

The Fc-GPVI-nt fusion protein is expressed in HELA cells using anadenoviral expression system to obtain soluble Fc-GPVI-nt.Characterization of the soluble forms of GPVI revealed that Fc-GPVI-ntis secreted as dimer with a molecular mass of approximately 160 kDa.Consistently, Miura and co-workers recently reported that GPVI-Fc-dimeris present as a dimer, in which two GPVI-Fc-dimer molecules arecross-linked by disulfide bonds formed from the Cys in the Fc domain ofeach molecule (21). Importantly, only the dimeric form of GPVI, but notmonomers of the extracellular domain of GPVI, has been reported toexhibit collagen binding affinity and to attenuate collagen-inducedplatelet aggregation (21).

Binding assays were performed to define GPVI-Fc-dimer-collageninteraction. Soluble GPVI binds to immobilized collagen in a saturablemanner. GPVI-Fc-dimer binding to fibrillar collagen was highly specific,since it did not occur to immobilized vWF or BSA. Further, GPVI bindingto immobilized collagen could be inhibited by soluble collagen. Highconcentrations of soluble collagen were required to block GPVI-Fc-dimerbinding, indicating the fusion protein binds immobilized collagen withhigh affinity. Correspondingly, a high association and dissociationconstant (K_(D) approximately 5.8×10⁻⁷ M) has been reported for theGPVI-collagen interaction (21).

Soluble Fc-GPVI-nt has been demonstrated earlier to attenuate plateletactivation and aggregation in response to collagen or convulxin, a snaketoxin, which binds to GPVI with high affinity (6;21;27). Apart fromplatelet aggregation, GPVI is critically involved in the process ofplatelet adhesion to collagen (3;22). In the present study, we,therefore, tested the effects of Fc-GPVI-nt on platelet adhesion underphysiological flow conditions in vitro. We show that soluble Fc-GPVI-ntdose-dependently inhibits platelet adhesion under low and high shearconditions in vitro. In the presence of Fc-GPVI-nt, but not of controlFc peptide, aggregation of adherent platelets was virtually absent,indicating that GPVI contributes to the processes of both plateletadhesion and subsequent activation by immobilized collagen. GPVI conferscollagen responses (i.e. adhesion and aggregation) in a receptordensity-dependent fashion (22). Correspondingly, it has been reportedthat a more than 50% reduction in GPVI expression transfected RBL-2H3cells is associated with a lack of collagen-induced aggregation in thesecells (8;22). Since a low variability in the GPVI receptor density hasbeen reported albeit in a small sample population (22), one might expectthat inhibition of approx. 50% of collagen-GPVI bonds is sufficient toattenuate platelet recruitment to exposed collagen. In the present studydoses of 1 mg/kg Fc-GPVI-nt were required to induce significantinhibition of platelet adhesion underflow, supporting the notion thatmultiple GPVI binding sites are available in each collagen fibril.Similar amounts of a function blocking anti-GPVI antibody were requiredto attenuate platelet-vessel wall injury in vivo (3).

Fibrillar collagen is a major constituent of the normal vessel wall butalso of atherosclerotic lesions (28). Rupture or fissuring of theatherosclerotic plaque results in exposure of collagen fibrils tocirculating platelets. As reported earlier, GPVI-collagen interactionsare essentially involved in arterial thrombus formation followingvascular injury (3). Here we demonstrate the in vivo effects of solubleFc-GPVI-nt on platelet recruitment after arterial injury. Endothelialdenudation was induced by reversible ligation of that carotid artery andthe dynamic process of platelet attachment was monitored by intravitalvideofluorescence microscopy as described (3). We demonstrate for thefirst time in vivo that soluble Fc-GPVI-nt attenuates stable platelettethering, adhesion and platelet aggregation following endothelialdenudation. Inhibition of platelet recruitment by Fc-GPVI-nt wasdose-dependent. Apart from preventing stable arrest of platelets,Fc-GPVI-nt significantly reduced initial platelet tethering/slow surfacetranslocation at sites of endothelial denudation. We have demonstratedearlier that inhibition of GPIba or of GPVI attenuate platelet tetheringto a similar extent (3), supporting that GPVI and GPIbα interaction needto act in contact to promote platlet tethering to subendothelialcollagen (2;29-31). In fact, the high “on”- and “off”-rates reported forthe GPVI-ligand interaction (22) are consistent with the role of GPVI asa tethering receptor.

The present invenvention identifies Fc-GPVI-nt as an active ingredientof a medicament to attenuate arterial thrombosis following vascularinjury. This concept is further supported by the observation thatFc-GPVI-nt is targeted to the exposed subendothelium at the site ofvascular injury, as demonstrated by immunohistochemistry. Thisimplicates that inhibition of GPVI-collagen interactions are likely tobe restricted to the site of vascular injury, while a prolonged systemicinhibition of platelet function is limited by the expected shorthalf-life of unbound Fc-GPVI-nt. In contrast, administration ofmonoclonal antibodies directed against GPVI inevitably leads to systemicinhibition of GPVI on all circulating platelets. In addition, Fc-GPVI-ntadministration did not affect platelet counts. In contrast, anti-GPVImAbs may eventually induce immune thrombocytopenia or a complete loss ofGPVI on circulating platelets (14;32), hampering their use in clinicalpractice. Accordingly, Fc-GPVI-nt therapy will likely be associated witha lower risk of clinical hemorrhage, compared to anti-GPVI mAb-basedstrategies.

Platelet adhesion and aggregation at sites of vascular injury is crucialfor hemostasis but may lead to arterial occlusion in the setting ofatherosclerosis and precipitate diseases such as coronary thrombosis andmyocardial infarction. The use of intravenous GPIIb-IIIa receptorinhibitors, has significantly improved the clinical success of patientsundergoing coronary stenting (33-35). However, severe bleedingcomplications have been reported to hamper the outcome of patientstreated with abciximab (36). The present invention demonstrates thatinhibition of GPVI-collagen interactions by Fc-GPVI-nt was sufficient tosignificantly reduce platelet adhesion both in vitro and in vivo;however, the soluble form of GPVI only moderately prolonged tailbleeding times. Similarly, mild bleeding disorders have been reported inpatients with GPVI-deficient platelets (37), indicating that coagulationand hemostasis are effective even in the complete absence of GPVI. Inpart this discrepancy may be due to the fact that inhibition or absenceof GPVI does not interfere with platelet aggregation in response toplatelet agonists other than collagen, e.g. ADP, tissue factor orthrombin. In contrast, direct inhibition of GPIIb-IIIa, e.g. by 7E3 orits humanized derivative, blocks fibrinogen binding to platelets, aprocess which is essential for platelet aggregation, and substantiallyattenuates platelet aggregation to most platelet agonist known thus far.Accordingly, Fc-GPVI-nt therapy are associated with a lower risk ofclinical hemorrhage, compared to anti-GPIIb-IIIa-based strategies.

In conclusion, the present invention provides the first in vivo evidencethat Fc-GPVI-nt attenuates platelet adhesion under flow in vitro andfollowing endothelial denudation in the carotid artery of mice in vivo.This further supports the concept that GPVI-collagen interactions play acentral role in all major phases of thrombus formation, i.e. platelettethering, firm adhesion, and aggregation at sites of arterial injury(e.g. during acute coronary syndromes). The present invention furthersupports the concept that GPVI plays a major role in the progression ofatherosclerosis. Moreover, the present invention shows for the firsttime the causal connection between GPVI and diabetes.

The invention will now be described in further detail with reference tothe following specific examples.

EXAMPLES

Animals. Specific pathogen-free C57BL6/J mice were obtained from CharlesRiver (Sulzfeld, Germany). For experiments, 12-weeks-old male mice wereused. All experimental procedures performed on animals were approved bythe German legislation on protection of animals.

Monoclonal antibodies. Monoclonal antibody (mAb) anti GPVI (JAQ1) andanti GPIbα (p0p/B) and Fab fragments from JAQ and p0p/B were generatedas described (Bergmeier, W., Rackebrandt, K., Schroder, W., Zirngibl, H.& Nieswandt, B. Structural and functional characterization of the mousevon Willebrand factor receptor GPIb-1× with novel monoclonal antibodies.Blood 2000; 95, 886-893; Nieswandt, B., Bergmeier, W., Rackebrandt, K.,Gessner, J. E. & Zirngibl, H. Identification of criticalantigen-specific mechanisms in the development of immunethrombocytopenic purpura in mice. Blood 2000; 96, 2520-2527). Irrelevantcontrol rat IgG was obtained from Pharmingen (Hamburg, Germany).

Generation of GPVI-Deficient Mice

To generate mice lacking GPVI, C57BL6/J wild-type mice were injectedwith 100 μg JAQ1 i.c. Animals were used for in vivo assessment ofplatelet adhesion on day 5 after mAb injection. Absence of GPVIexpression on platelets was verified by Western blot analysis and flowcytometry.

Flow Cytometry

Heparinized whole blood, obtained from wild type C57BL6/J mice orGPVI-depleted mice was diluted 1:30 with modified Tyrodes-HEPES buffer(134 mM NaCl, 0.34 mM Na₂HPO₄, 2.9 mM KCl, 12 mM NaHCO₃, 20 mM HEPES, 5mM glucose, and 1mM MgCl₂₁ pH 6.6). The samples were incubated withfluorophore-labeled mAb anti-GPVI (JAQ1) and anti-CD41 for 10 min atroom temperature and directly analyzed on a FACScan™ (Becton Dickinson).

Cloning, viral expression and purification of soluble human and murineGPVI. To generate a soluble form of human GPVI, the extracellular domainof human GPVI was cloned and fused to the human immunoglobin Fc domainaccording to the following examples 1 to 3. Adenoviral constructs codingfor the GPVI-Fc-fusion protein or control Fc were prepared to generatethe recombinant protein. GPVI-Fc and control Fc were expressed assecreted soluble proteins using the human HELA cell line to preventmisfolding and non-glycosylation of the expressed proteins.

Example 1 Cloning of the Immunoadhesin of GP VI (Fc-GPVI-nt)

We generated an immunoadhesin of the GP VI receptor by generating arecombinant fusion protein of the n-terminal part of GP VI —whichencodes the extracellular domain of GPVI-together with the Fc part of anIgG. The Fc was amplified from a human heart cDNA library (Clonetech,Palo Alto, Calif.) by PCR using the forward primer5′-cgcggggcggccgcgagt-ccaaatcttgtgacaaaac-3′ and the reverse primer5′-gcgggaagctttcatttacccggagacagggag-3′. The PCR reaction was performedat 58° C. annealing temperature and 20 cycles with the Expand HighFidelity PCR Sytem (Roche Molecular Biochemicals, Mannheim, Germany).The PCR fragment was cloned in the plasmid pADTrack CMV withNotI/HindIII and the sequence was checked by sequencing (MediGenomix,Martinsried, Germany).

For cloning of the extracellular domain of the human GPVI RNA fromcultured megakaryocytes was isolated (RNeasy Mini Kit; Qiagen, Hilden,Germany) according to the manufacters protocol and reverse transcriptionwas performed (Omniscript RT Kit; Qiagen) with 2 μg RNA at 37° C.overnight. 100 ng of the reaction was used as a template in PCRamplification of the hGPVI with the primer5′-gcggggagatctaccaccatgtctccatccccgacc-3′ and5′-cgcggggcggccgccgttgcccttggtgtagtac-3′. The PCR reaction was performedat 54° C. annealing temperature and 24 cycles with the Expand HighFidelity PCR Sytem (Roche Molecular Biochemicals, Mannheim, Germany).The PCR fragment was cloned in the plasmid pDATrack CMV Fc withBgIII/NotI and the sequence was checked by sequencing.

Construction of a Monomeric Fusion Protein Based on Fc-GPVI-Nt

The Fc monomer fragment was amplified by PCR using the primer pair5′-cgcggggcggccgcccagcacctgaactcctg-3′ and5′-cgcggggatatctcatttacccggagacagggag-3′ and pADTrack CMV gpVI-Fc as atemplate. The PCR reaction was performed at 58° C. annealing temperatureand 20 cycles with the Expand High Fidelity PCR Sytem (Roche MolecularBiochemicals, Mannheim, Germany). The Fc monomer PCR fragment(NotI/EcoRV) and the gpVI fragment from pADTrack CMV gpVI-Fc(BgIII/NotI) were cloned as described above.

Example 2 Generation of the Adenovirus for Fc-GPVI-nt (Ad-Fc-GPVI-nt)

The plasmid pADTrack CMV Fc-GPVI-nt was linearized with Pmel (NewEngland Biolabs, Beverly, Mass.) overnight, dephosphorylated andpurified (GFX DNA and Gel Purification Kit; Amersham Pharmacia Biotech,Uppsala, Sweden). For recombination electrocompetent E. coli BJ5183(Stratagene, La Jolla, Calif.) were cotransformed with 1 μg of thelinearized plasmid and 0.1 μg pAdeasy1 at 2500 V, 200 Ω and 25 μFD (E.coli-pulser; Biorad, Heidelberg, Germany), plated and incubatedovernight at 37° C. The colonies were checked after minipreparation ofthe plasmid-DNA with Pacl and the positive clones were retransformed inE. coli DH5α.

For transfection (Effectene Transfection reagent; Qiagen, Hilden,Germany) of 293 cells plasmid-DNA was digested with Pacl. The cells werecultured for 7 days and harvested by scraping and centrifugation. Thepellet was resuspended in Dulbecco's PBS and the cells were lysed byfour repetitive freezing (−80° C.) and thawing (37° C.) cycles. Celldebris was removed by centrifugation and the lysate stored at −80° C.

For plaque selection of recombinant virus 293 cells are infected inDulbeccos PBS for 1 hour at room temperature under gentle agitation withdifferent serial dilutions of lysate from transfection. Following theinfection, the cells are overlayed with growth medium containing 0.5%agarose (1:1 mix of modified Eagles medium 2×, Gibco Life Technologies#21935, supplemented with 20% serum, 2×Pencillin/Streptomycin,2×L-glutamin and agarose in water 1%, Seacam). 5-14 days post infectionthe cell layer was monitored for formation of plaques which were pickedusing a pasteur pipett, resuspended in 0.5 ml Dulbeccos PBS and storedat −80° C. The plaques were used for further amplification rounds on 293cells.

Construction of Human gpVI-Fc Monomer Expressing Stable CHO

The monomer expressing cells were generated in accordance with examle 2.

Example 3 Fc-GPVI-nt Protein and Fc Control Immunoadhesin Purification

The culture supernatant of Ad-Fc-GPVI-nt-infected Hela cells wascollected 2 days after infection, centrifugated (3800 g, 30 min, 4° C.)and filtrated (0.45 μm). The immunoadhesin was precipitated by additionof 1 vol. ammonium sulfate (761 g/l) and stirred overnight at 4° C. Theproteins were pelleted by centrifugation (3000 g, 30 min, 4° C.),dissolved in 0.1 Vol PBS and dialysed in PBS overnight at 4° C. Theprotein solution was clarified by centrifugation (3000 g, 30 min, 4° C.)and loaded on a protein A column (HiTrap™ protein A HP, AmershamPharmacia Biotech AB, Uppsala, Sweden). The column was washed withbinding buffer (20 mM sodium phoshate buffer pH 7.0, 0.02% NaN₃) untilOD₂₈₀<0.01 and eluted with elution buffer (100 mM glycine pH 2.7). Theeluted fractions were neutralized with neutralisation buffer (1 MTris/HCl pH 9.0, 0.02% NaN₃), pooled, dialysed in PBS overnight at 4°C., aliquotated and frozen at −20° C. The molecular mass of Fc-GPVI-ntprotein was −80 kDa under reducing conditions in SDS-PAGE, as detectedby Coomassie blue stain or by immunoblotting with peroxidase-conjugatedgoat anti-human Fc antibody or by the anti-GPVI mAb 5C4 (FIG. 1 a, upperand middle panel). In contrast, a −160 kDa protein was identified undernon-reducing conditions (FIG. 1 a, lower panel), supporting the notionthat GPVI-Fc is obtained solely as dimer (21).

Example 4 GP VI Inhibitor Screening Assay

ELISA plates (Immulon2 HB, Dynx Technologies, Chantilly, Va.) werecoated overnight at 4° C. with 1 μg/well collagen (type I bovine; BDBioscience, Bedford, Mass.) in 100 μl 50 mM Tris/HCl pH 8.0. The platewas washed with 250 μl/well PBS/0.05% Tween 20 (PBST) twice and blockedwith 250 μl/well Roti-Block (Roth, Karlsruhe, Germany) overnight. Theplate was washed with 250 μl/well PBST twice, 100 μl Fc-GPVI-nt in PBSTwas added (optimal 2 μg/well) and the plate was incubated for 1 h atroom temperature. After 5-fold washing with 250 μl PBST 100 μlperoxidase-conjugated goat anti-human IgG antibody (Dianova, Hamburg,Germany) was added in a dilution of 1:10000 and incubated for 1 h atroom temperature. After repeated washing with 250 pI PBST 100 μldetection reagent (BM Blue POD Substrate; Roche, Mannheim, Germany) wasadded and incubated for 15 min. The reaction was stopped by the additionof 100 μl 1 M H₂SO₄ and the plate was measured at 450 nm against thereference wavelength 690 nm. To screen for potential inhibitors, testcompounds are added to the incubation in 100 μl PBST at variousconcentrations.

Example 5 Platelet Aggregation and Luminometry

Platelet aggregation ex vivo and in vitro was evaluated by opticalaggregometry in citrated blood samples at 370° C. using a two channelChronolog aggregometer (Nobis, Germany). Platelet-rich plasma wasprepared from citrated whole blood by centrifugation (200 g for 20 min).The final platelet count was adjusted to 2×1 0 platelets/ml withautologous plasma. After adjustment of the baseline, collagen (type 1,bovine) from 0.2 to 4 μg/ml was added and aggregation was recorded for 5min. Simultaneously, release of ATP was recorded using the fireflyluminometer method. Incubation with the monoclonal GP VI antibody JAQ 1was performed for 15 min with 50 μg/ml antibody.

Example 6 In Vitro Platelet Adhesion Assay for GP VI/CollagenInteraction

From ACD (20% final concentration) blood platelet rich plasma wasprepared and adjusted to a final concentration of 10⁸ platelets/ml byHepes Tyrode (pH 6.5). Coverslips were coated with monolayers of variousadhesive proteins (Collagen, vWF) at different concentrations. Perfusionstudies were carried out in a perfusion chamber generated from glasscoverslips. Perfusion was performed at shear rates of 500/s representinglow-medium flow and 2000/s representing high shear rates. Adhesion wasmeasured at 37° C. for 20 minutes and then drawn through the chamber atfixed wall shear rates for 5 minutes using an automated syringe pump.After perfusion the coverslips were gently washed with Hepes Tyrode,taken from the chamber. Coverslips were repeatedly washed with HepesTyrode to completely remove adhesive platelets. The platelets insuspension were quantitatively analysed by FACS measurements. Theanalysis of the functional status of platelets was further assessed byanalysis of surface marker expression (CD 41; CD 61 and CD 62 P)according to the standard flow cytometry protocol.

Example 7 Preparation of Platelets for Intravital Microscopy

Platelets (wild type, or GPVI-deficient) were isolated from whole bloodas described (Massberg, S. et al. Platelet-endothelial cell interactionsduring ischemia/reperfusion: the role of P-selectin. Blood 1998; 92,507-515) and labeled with 5-carboxyfluorescein diacetat succinimidylester (DCF). The DCF-labeled platelet suspension was adjusted to a finalconcentration of 200×10⁶ platelets/250 μl. Where indicated, fluorescentwild type platelets were preincubated with 50 μg/ml anti-GPVI (JAQ1) Fabfragments, or anti GPIbα (p0p/B) Fab fragments for 10 min. Subsequently,the pretreated platelets together with the Fab fragments were infusedinto wild type recipient mice and platelet adhesion was assessed priorto and after carotid injury by in vivo video microscopy, as describedbelow.

Example 8 Assessment of Platelet Adhesion and Aggregation by IntravitalMicroscopy

Wild type C57BL6/J or GPVI-deficient mice were anesthetized byintraperitoneal injection of a solution of midazolame (5 mg/kg bodyweight, Ratiopharm, Ulm, Germany), medetomidine (0.5 mg/kg body weight,Pfizer, Karlsruhe, Germany), and fentanyl (0.05 mg/kg body weight,CuraMed Pharma GmbH, Munich, Germany). Polyethylene catheters (Portex,Hythe, England) were implanted into the right jugular vein andfluorescent platelets (200×10⁶/250 μl) were infused intravenously. Theright common carotid artery was dissected free and ligated vigorouslynear the carotid bifurcation for 5 min to induce vascular injury. Priorto and following vascular injury, the fluorescent platelets werevisualized in situ by in vivo video microscopy of the right commoncarotid artery. Platelet-vessel wall interactions were monitored using aZeiss Axiotech microscope (20× water immersion objective, W 20×/0.5,Zeiss) with a 100 W HBO mercury lamp for epi-illumination. Allvideo-taped images were evaluated using a computer-assisted imageanalysis program (Cap Image 7.4, Dr. Zeintl, Heidelberg, Germany).Transiently adherent platelets were defined as cells crossing animaginary perpendicular through the vessel at a velocity significantlylower than the centerline velocity; their numbers are given as cells permm² endothelial surface. The number of adherent platelets was assessedby counting the cells that did not move or detach from the endothelialsurface within 10 seconds. The number of platelet aggregates at the siteof vascular injury was also quantified and is presented per mm².

Example 9 Scanning Electron Microscopy

Following intravital videofluorescence microscopy, the carotid arterywas perfused with PBS (37° C.) for 1 min, followed by perfusion fixationwith phosphate-buffered glutaraldehyde (1% vol/vol). The carotid arterywas excised, opened longitudinally, further fixed by immersion in 1%PBS-buffered glutaraldehyde for 12 hours, dehydrated in ethanol, andprocessed by critical point drying with CO₂. Subsequently, the carotidartery specimens were oriented with the lumen exposed, mounted withcarbon paint, sputter coated with platinum, and examined using a fieldemission scanning electron microscope (JSM-6300F, Jeol Ltd., Tokyo,Japan).

Example 10

Assessment of Fc-GPVI-nt binding to immobilized collagen. The binding ofFc-GPVI-nt to immobilized collagen was determined. ELISA plates(Immulon2 HB, Dynx Technologies, Chantilly, Va.) were coated over nightat 4° C. with 1 μg collagen (typl bovine; BD Bioscience, Bedford, Mass.)in 100 μl coating buffer (1.59 g/l Na₂CO₃, 2.93 g/l NaHCO₃, 0.2 g/lNaN₃, pH 9.6). The plates were washed with 250 pl/well PBS/0.05% Tween20 (PBST) twice and blocked with 250 μl/well Roti-Block (Roth,Karlsruhe, Germany) over night. The plates were washed with 250 μl/wellPBST twice, then 3.0, 6.0, 12.5, 25.0, 50.0 or 100 μg/ml Fc-GPVI-nt inPBST was added and the plate was incubated for 1 hr at room temperature.Where indicated, Fc-GPVI-nt (20 μg/ml) was preincubated for 10 min withsoluble collagen. After incubation the plates were washed 5 times with250 μl PBST and peroxidase-conjugated goat anti-human IgG antibody Fcγfragment specific (109-035-098; Dianova, Hamburg, Germany) was added ina dilution of 1:10.000 and incubated for 1 hr at room temperature. After5 fold washing with 250 μl PBST 100 μl detection reagent (BM Blue PODSubstrate; Roche, Mannheim, Germany) was added and incubated up to 10min. The reaction was stopped by the addition of 100 μl 1 M H₂SO₄ andthe plate was measured at 450 nm against reference wavelength 690 nm.

Fc-GPVI-nt showed a dose-dependent and saturable binding to immobilizedcollagen (FIG. 9 b). Half maximal collagen binding was observed at afinal Fc-GPVI-nt concentration of 6.0 μg/ml. Binding of GPVI-Fc did notoccur to BSA, vWF (FIG. 9 c, left panel) or Poly-L-Lysin (not shown),supporting the specificity of Fc-GPVI-nt binding. Moreover, we did notdetect any significant binding of the control Fc protein lacking theexternal GPVI domain under identical conditions (FIG. 9 c, right panel).

To further address the specificity of GPVI-binding, we the ability ofsolubilized fibrillar collagen to compete with immobilized collagen forthe association with Fc-GPVI-nt was tested. Soluble collagen inhibitedFc-GPVI-nt-binding to immobilized collagen in a dose-dependent manner(FIG. 9 d). A concentration of 100 μg/ml soluble collagen was requiredto reduce Fc-GPVI-nt binding by more than 50%. Together, these dataindicated that Fc-GPVI-nt binding to collagen is specific andcharacterized by high affinity.

Example 11

Generation of monoclonal antibody against human GPVI. Monoclonalantibodies were generated essentially as described (17). Lou/C rats wereimmunized with the adenovirally expressed human Fc-GPVI-nt fusionprotein. Screening of hybridoma supernatants was performed in asolid-phase immunoassay using Fc-GPVI-nt or FC lacking the GPVI domain.Screening identified the supernatant of hybridoma 5C4 to bindspecifically to Fc-GPVI-nt but not to Fc lacking the external GPVIdomain. The immunoglobulin type was determined with rat Ig class(anti-IgM) and IgG subclass-specific mouse mAbs. The monoclonalantibodies were purified using Protein G-Sepharose columns. Antibodyspecificity of 5C4 was verified by immunoblotting against Fc-GPVI-nt andcontrol Fc. 5C4 monoclonal antibody detected adenovirally expressedFc-GPVI-nt but not control Fc. Furthermore, human GPVI was recovered inlysates obtained from human platelets. In addition, 5C4 bindsspecifically to the surface of platelets but not of leukocytes or redblood cells, as demonstrated using flow cytometry (not shown).

Example 12

FACS measurement of CD62 P externalisation. Human citrate blood wascollected from volunteers. Platelet rich plasma (PRP) was generatedafter centrifugation and washing procedures (PBS 1×; pH 7.2) with 2000rpm at 4° C. and resuspension. PRP diluted in staining buffer (1×PBS(w/o Ca²⁺ and Mg⁺) with 0.1% sodium azide and 2% fetal bovine serum (FBS2 mM CaCl) was incubated with equine collagen type 1 (0; 2; 5 and 10μg/ml; Nobis) in the presence of Fc-GPVI-nt (100 μg/ml) or equimolarconcentrations control Fc. Anti CD 62P antibodies labelled with thefluorophor peroxidase (Immunotech) were added. FACS measurement wasperformed with an Becton Dickenson FACScalibur device.

Increasing concentrations of collagen led to platelet secretion fromalpha granules indicated by CD 62P externalisation. Co-incubation ofcollagen with Fc-GPVI-nt blunted the CD62 P externalisation determinedby FACS (FIG. 10).

Example 13

Platelet aggregation and ATP release. PRP was generated as describedabove. Aggregation was determined in a Whole-Blood-Aggregometer 500VS(Chrono-Log Corporation). Platelet cell number from PRP was adjusted to1.0×10⁸ cells/ml by Thyrodes-HEPES buffer (2.5 mmol/l HEPES, 150 mmol/lNaCl, 12 mmol/l NaHCO₃, 2,5 mmol/l KCl, 1 mmol/l MgCl₂, 2 mmol/l CaCl₂,5,5 mmol D-Glucose, 1 mg/ml BSA, pH 7.4). Chrono-Lume #395 (Chrono-LogCorporation) was added for ATP measurement. Agonists were added to theplatelets, pipetted into the aggregometer and aggregation was startedunder defined stirring conditions. Aggregation was determined by changeof light transmission due to coagulating platelets and normalised to aninternal standard. ATP release is determined at the characteristicwavelength of Chrono-Lume for ATP and normalised to an internal standardaccording to the manufacturer's instructions.

Platelet aggregation and ATP release was specifically inhibited byFc-GPVI-nt for collagen mediated agonist stimulation (FIG. 11 a & b).ADP— and thrombin-mediated (TRAP 10 μM) platelet aggregation and ATPrelease was unaffected by Fc-GPVI-nt.

Example 14

PDGF release from human platelets. PRP from human volunteers wasprepared as described above. PDGF release from human platelets wasdetermined with a kit system (R & D Systems # DHD00B) according to themanufacturer's instructions. PDGF release was stimulated with collagentype 1 (20 μg/ml; Nobis) under control conditions and in the presence ofFc-GPVI-nt (100 μg/ml) or equimolar concentrations of control Fc. PDGFrelease is normalised to the manufacturer's standard probe.

PDGF release as an indicator for release of endogenous transmitters fromalpha granules of platelets was also blunted after collagen stimulation.(FIG. 1 c).

Example 15

Effect of Fc-GPVI-nt on bleeding time from human whole blood in vitro.In vitro bleeding time was determined with an PFA-100 device(Dade-Behring). 800 μl of human whole blood was injected in the PFA-100device. Bleeding time was measured with ADP/collagen andepinephrine/collagen coated measuring cells according to themanusfacturer's instructions.

There was no significant prolongation of bleeding time in vitro (PFA-100device) with increasing concentrations of Fc-GPVI-nt after differentagonist stimulations. In contrast, therapeutically relevantconcentrations of ReoPro maximally prolonged bleeding time in thePFA-100 device (FIG. 12).

Example 16

Effect of soluble GPVI on platelet adhesion to immobilized collagenunder flow. Human platelets were isolated from ADC-anticoagulated wholeblood as described (18). Washed platelets were resuspended inTyrodes-HEPES buffer (2.5 mmol/l HEPES, 150 mmol/l NaCl, 12 mmol/lNaHCO₃, 2,5 mmol/l KCl, ₁ mmol/l MgCl₂, 2 mmol/l CaCl₂, 5,5 mmolD-Glucose, 1 mg/ml BSA, pH 7.4) to obtain a platelet count of 2×10⁸cells/ml. Adhesion of platelets to plates coated with immobilizedcollagen was determined in a parallel plate flow chamber in the presenceof 200 μg/ml Fc-GPVI-nt or control Fc.

GPVI plays a crucial role in the process of platelet recruitment toimmobilized collagen in vitro (22). We determined the effect ofFc-GPVI-nt on adhesion of human platelets to immobilized collagen undershear conditions in vitro. As reported by others earlier (23), plateletsadhered firmly to immobilized collagen at both low (500 sec⁻¹) and high(1000 sec¹) shear rates forming thrombi (FIG. 13). Soluble Fc-GPVI-nt,but not control Fc lacking the external GPVI domain, significantlyattenuated platelet adhesion on immobilized collagen by 37 and 44% atshear rates of 500 sec⁻¹ and 1000 sec⁻¹, respectively (FIG. 13).Inhibition was specific since Fc-GPVI-nt did not affect plateletadhesion to immobilized vWF.

Example 17

Determination of Fc-GPVI-nt plasma concentrations was carried out withan IMMUNO-TEK ELISA system for the quatitative determination of humanIgG (ZeptoMetrix Corporation; Cat # 0801182). Specific peroxidaseconjugated goat anti-human IgG antibodies against the Fc part of theFc-GPVI-nt are used (Dianova). After several washing steps with PBS-Taccording to the manufacturer's specifications peroxidase substrate (BMBlue POD, Roche) is added and measured at the characteristic 450 nmwavelength in an ELISA assay reader (Tecan Sunrise). The Fc-GPVI-ntconcentration is quantified by comparison to an internal human IgGstandard. Fc-GPVI-nt showed favourable in vivo pharmacokinetics. Aftersingle intraperitoneal injection in mice high plasma levels weremeasurable after 24 hours and the half life of the fusion proteinexceeded 96 hours (FIG. 14 a). Repeated intraperitoneal injection wasleading to blood accumulation of the fusion protein (FIG. 14 b)suggesting favourable kinetics for long term application for thetreatment of chronic diseases. After single intravenous injection ofFc-GPVI-nt with increasing doses, dose-dependent plasma concentrationsof Fc-GPVI-nt were detectable over 5 to 60 minutes up to 14 hours (FIG.14 c).

Example 18

Preparation of murine platelets for intravital fluorescence microscopy.Murine platelets were isolated from whole blood and labeled with5-carboxyfluorescein diacetate succinimidyl ester (DCF) as reportedearlier (19). The DCF-labeled platelet suspension was adjusted to afinal concentration of 200×10⁶ platelets/250 μl. Adhesion of murineplatelets was assessed prior to and after carotid injury by in vivovideo microscopy, as described below.

Example 19

Carotid ligation and assessment of platelet adhesion and aggregation byintravital microscopy. Platelet recruitment following endothelialdenudation was performed as reported earlier (3). In brief, wild typeC57BL6/J mice were anesthetized by intraperitoneal injection of asolution of midazolame (5 mg/kg body weight, Ratiopharm, Ulm, Germany),medetomidine (0.5 mg/kg body weight, Pfizer, Karlsruhe, Germany), andfentanyl (0.05 mg/kg body weight, CuraMed Pharma GmbH, Munich, Germany).Where indicated, Fc-GPVI-nt (1 or 2 mg/kg body weight) or control Fc inan amount equimolar to 2 mg/kg Fc-GPVI-nt was administeredintravenously. Thereafter, endothelial denudation was induced near thecarotid bifurcation by vigorous ligation for 5 min. Following inductionof vascular injury luorescent platelets (200×10⁶/250 μl) were infusedintravenously via polyethylene catheters (Portex, Hythe, England)implanted into the right jugular vein. The fluorescent platelets werevisualized in situ by in vivo video microscopy of the right commoncarotid artery using a Zeiss Axiotech microscope (20× water immersionobjective, W 20×/0.5, Zeiss) with a 100 W HBO mercury lamp forepi-illumination. All video-taped images were evaluated using acomputer-assisted image analysis program (Cap Image 7.4, Dr. Zeintl,Heidelberg, Germany (19;20)). Tethered platelets were defined as allcells establishing initial contact with the vessel wall, followed byslow surface translocation (at a velocity significantly lower than thecenterline velocity) or by firm adhesion; their numbers are given ascells per mm² endothelial surface. The number of adherent platelets wasassessed by counting the cells that did not move or detach from theendothelial surface within 10 seconds. The number of platelet aggregatesat the site of vascular injury was also quantified and is presented permm². In addition, the total thrombus area was assessed using Cap Image7.4.

Example 20

Scanning electron microscopy. Following intravital videofluorescencemicroscopy, the carotid artery was perfused with PBS (37° C.) for 1 minin three animals per group, followed by perfusion fixation withphosphate-buffered glutaraldehyde (1% vol/vol). The carotid artery wasexcised, opened longitudinally, further fixed by immersion in 1%PBS-buffered glutaraldehyde for 12 hours, dehydrated in ethanol, andprocessed by critical point drying with CO₂. Subsequently, the carotidartery specimens were oriented with the lumen exposed, mounted withcarbon paint, sputter coated with platinum, and examined using a fieldemission scanning electron microscope (JSM-6300F, Jeol Ltd., Tokyo,Japan).

Example 21

Assessment of in vivo Fc-GPVI-nt binding by immunohistochemistry.Carotid arteries obtained from mice treated with Fc-GPVI-nt were shockfrozen and embedded in cryoblocks (medite, Medizintechnik GmbH,Burgdorf, Germany). The binding of Fc-GPVI-nt to the endothelium andsubendothelium was determined on 5 μm cryostat sections, stained withperoxidase-conjugated goat anti-human IgG antibody Fcy fragment specific(109-035-098; Dianova, Hamburg, Germany). Carotid arteries obtained fromFc-treated mice served as controls.

Example 22

Effect of soluble GPVI on platelet counts, bleeding time and plateletadhesion in vivo. Animals were treated with 2 mg/kg or 4 mg/kgFc-GPVI-nt or equimolar doses of control Fc lacking the external GPVIdomain. Infusion of Fc-GPVI-nt or control Fc even at the highest dose of4 mg/kg had not significant effects on peripheral platelet counts.Moreover, the Fc-GPVI-nt fusion protein, did not induce any significantprolongation of tail bleeding times compared to control animals (FIG. 15a). The absolute bleeding times were 1.9±0.9 in PBS treated mice and2.9±1.9 min and 4.6±0.6 min in mice treated with 2 mg/kg or 4 mg/kgFc-GPVI-nt. In contrast, bleeding times were prolonged condsiderably(42.6±21.6) in Integrilin-treated animals (0.2 mg per kg IV).

The effects of Fc-GPVI-nt on platelet recruitment in a mouse model ofcarotid injury may be studied using intravital fluorescence microscopy.Animals were treated with 1 mg/kg or 2 mg/kg Fc-GPVI-nt or an equimolaramount of control Fc lacking the external GPVI domain as describedabove. After infusion of Fc-GPVI-nt or control Fc endothelial denudationof the mouse carotid artery was induced by vigorous ligation as reportedpreviously (3). Ligation of the carotid artery consistently causedcomplete loss of the endothelial cell layer. Platelet adhesion wasdirectly visualized and quantified using in vivo fluorescence microscopy(19;20) (FIG. 15 d). In control (Fc-treated) mice numerous plateletswere tethered to the vascular wall within the first minutes afterendothelial denudation (12.026±1.115 tethered platelets/mm²). Plateletsestablishing contact with the subendothelium exhibited initially a slowsurface translocation, which is frequently followed by subsequent firmplatelet adhesion and platelet aggregation (5.494±874 adherentplatelets/mm² and 114±17 platelet thrombi/mm²). In contrast, in thepresence of Fc-GPVI-nt platelet recruitment to the site of vascularinjury was dramatically attenuated. Platelet tethering was reduced by 65and 71% compared to Fc-treated animals following pretreatment with 1mg/kg or 2 mg/kg Fc-GPVI-nt (P<0.05 vs. control). In parallel, firmplatelet adhesion was reduced in a dose-dependent manner (by 49 and 65%following administration of 1 mg/kg or 2 mg/kg Fc-GPVI-nt, respectively;P<0.05 vs. control). Likewise, aggregation of adherent platelets wasvirtually absent in animals treated with 2 mg/kg Fc-GPVI-nt fusionprotein (P<0.05 vs. control Fc, FIG. 15 b-d). Scanning electronmicroscopy also clearly demonstrated that platelet adhesion andaggregation following endothelial denudation of the common carotidartery were virtually absent in Fc-GPVI-nt treated, but not inFC-pretreated mice (FIG. 15 e). To confirm the presence of Fc-GPVI-nt atthe site of injury, the carotid arteries were excised following in vivomicroscopy and processed further for immunohistochemistry usingperoxidase-conjugated goat anti-human IgG antibodies. InFc-GPVI-nt-treated mice Fc-GPVI-nt was detected on at the luminal aspectof the site of vascular damage (FIG. 15 f). Together, these datademonstrate that Fc-GPVI-nt specifically binds to sites of vascularinjury in vivo and prevents subsequent platelet recruitment.

Effect of soluble GPVI on atherosclerosis. 4 weeks old apoE −/− mice(The Jackson Laboratory) consumed a 0.25% cholesterol diet (HarlanResearch diets) for 6 weeks. After 2 weeks 4 apoE −/− mice were injectedwith Fc-GPVI-nt 200 μg per mouse twice weekly with continous cholesteroldiet. 4 apoE −/− mice with the similar protocol were injected with thecontrol Fc protein (200 μg) twice weekly and served as control mice. Forassessment of plaque formation the animals were killed and the vasculartree was carefully dissected from the animals. The whole preparations ofthe aortae and carotides were flushed with 0.9% sodium chloride andfixed. The complete vascular preparation was stained with SUDAN III redto assess plaque formation and viewed under a microscope. Treatment ofatherosclerosis prone apoE −/− knockout mice with Fc-GPVI-nt over 4weeks significantly attenuated atheroprogression. (FIG. 16).

Example 23

FACS measurement of CD61 and CD32 surface expression on platelets fromdiabetic patients. Human citrate blood was collected from 111 patientssuffering from diabetes or from 363 non-diabetic patients. Platelet richplasma (PRP) was generated after centrifugation and washing procedures(PBS 1x, pH 7.2) with 2000 rpm at 4° C. and resuspension. Anti CD61 andanti CD32 antibodies labelled with the fluorophor peroxidase(Immunotech) were added or or the anti monoclonal anti-GPVI antibody 4C9labelled with FITC. FACS measurement was performed with an BectonDickenson FACScalibur device. Surface expression was quantified byfluorescence. Correleation of CD32 fluorescence and 4C9 fluorescence wascalculated with the correlation coefficient r=0.516.

Statistical Analysis. Comparisons between group means were performedusing Mann-Whitney Rank Sum Test. Data represent mean±s.e.m. A value ofP<0.05 was regarded as significant.

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1-25. (canceled)
 26. A fusion protein comprising: a) the extracellulardomain of GPVI; b) a linker comprising the amino acids Gly-Gly-Arg; andc) the Fc domain of an immunoglobulin.
 27. The fusion protein of claim26, wherein the extracellular domain of GPVI comprises amino acids 1-267of SEQ ID NO:
 1. 28. The fusion protein of claim 26, wherein the Fcdomain of the immunoglobulin comprises amino acids 273-504 of SEQ IDNO:
 1. 29. A homodimer comprising the fusion protein of claim
 26. 30. Afusion protein comprising: a) the extracellular domain of GPVI; b) alinker comprising from 1-100 amino acids with the proviso that thelinker is not three amino acids; and c) the Fc domain of animmunoglobulin.
 31. The fusion protein of claim 30, wherein the linkercomprises from 1-10 amino acids.
 32. The fusion protein of claim 30,wherein the extracellular domain of GPVI comprises amino acids 1-267 ofSEQ ID NO:1.
 33. The fusion protein of claim 30, wherein the Fc domainof the immunoglobulin comprises amino acids 273-504 of SEQ ID NO:
 1. 34.A homodimer comprising the fusion protein of claim
 30. 35. Animmunoadhesin comprising: a) the extracellular domain of GPVI; b) alinker comprising the amino acids Gly-Gly-Arg; and c) the Fc domain ofan immunoglobulin.
 36. The immunoadhesin of claim 35, wherein theextracellular domain of GPVI comprises amino acids 1-267 of SEQ IDNO:
 1. 37. The immunoadhesin of claim 35, wherein the Fc domain of theimmunoglobulin comprises amino acids 273-504 of SEQ ID NO:
 1. 38. Afusion protein/immunoadhesin comprising the amino acid sequence of SEQID NO:1.
 39. An isolated nucleic acid encoding the fusion protein orimmunoadhesin of claim
 26. 40. An isolated nucleic acid comprising thenucleotide sequence of SEQ ID NO:2.
 41. A method of inhibitingligand-mediated GPVI activation, comprising contacting the fusionprotein of claim 1 with collagen under conditions whereby thecomposition binds collagen, thereby inhibiting ligand-mediated GPVIactivation.
 42. A method of blocking the binding of GPVI with collagen,comprising contacting collagen with the fusion protein of claim 1 underconditions whereby the composition binds collagen, thereby blocking thebinding of GPVI to collagen.
 43. A method of preventing binding ofplatelets to blood-exposed collagen in the vascular system, comprisingcontacting said collagen with the fusion protein of claim
 1. 44. Acomposition comprising the fusion protein of claim 1 in apharmaceutically acceptable carrier.